low-frequency stimulation induces a new form of ltp, metabotropic glutamate (mglu5) receptor- and...

Low-Frequency Stimulation Induces a New Form of LTP, MetabotropicGlutamate (mGlu5) Receptor- and PKA-Dependent, in the CA1 Area

of the Rat Hippocampus

Fabien Lante, Marie-Celeste de Jesus Ferreira, Janique Guiramand, Max Recasens,and Michel Vignes*

ABSTRACT: Low frequency-induced short-term synaptic plasticity wasinvestigated in hippocampal slices with 60-electrode recording array.Remarkably, the application of low-frequency stimulation (1 Hz) for ashort duration (3–5 min) resulted in the induction of a slow-onset long-term potentiation (LTP) in the immediate vicinity of the stimulated elec-trode. This phenomenon was observed exclusively in the CA1 subfield,neither in the CA3 area nor in the dentate gyrus. The induction of thisslow-onset LTP required neither N-methyl-D-aspartate (NMDA) nor non-NMDA ionotropic receptor activation but was strongly dependent onmetabotropic glutamate mGlu5 receptor stimulation and [Ca2þ]i in-crease. In addition, this form of synaptic plasticity was associated withan increase in cAMP concentration and required protein kinase A acti-vation. Paired-pulse facilitation ratio and presynaptic fiber volley ampli-tude were unaffected when this LTP was triggered, suggesting the in-volvement of postsynaptic modifications. Although mitogen activatedprotein kinase pathway was stimulated after the application of low fre-quency, the induction and maintenance of this slow-onset LTP were notdependent on the activation of this intracellular pathway. The directactivation of adenylyl cyclase with forskolin also induced a synapticenhancement displaying similar features. This new form of LTP couldrepresent the mnesic engram of mild and repetitive stimulation involvedin latent learning. VVC 2005 Wiley-Liss, Inc.

KEY WORDS: glutamate; hippocampal slice; MAP kinase; micro-electrode array; plasticity

INTRODUCTION

Activity-dependent alterations of excitatory synaptic transmission areprevalent mechanisms of synaptic plasticity underlying learning and mem-ory processes in the mammalian brain. Excitatory synapses are continuallyadapting their strength—either increasing or decreasing—for various dura-tions, according to the pattern of afferent stimulation. This is particularlyrelevant and well-documented in the hippocampus (Bliss and Colling-ridge, 1993; Nicoll and Malenka, 1995). Brief high-frequency stimulation(HFS; 100 Hz) generally produces long-lasting enhancement of synapticefficacy (long-term potentiation, LTP). By contrast, prolonged low-fre-quency stimulation (LFS; 1 Hz) mediates long-lasting depression of syn-

aptic transmission (long-term depression, LTD). Theduration of LFS protocols appears to be crucial todetermine the direction of the synaptic modification.Indeed, while long-lasting LFS (15 min) leads to LTD(Kemp and Bashir, 2001), short-lasting LFS protocols(5 min) may be associated with short-term synapticfacilitation (Salin et al., 1996). These activity-depend-ent phenomena appear to be region-specific in the hip-pocampus: HFS-mediated LTP is either dependent onor independent of N-methyl-D-aspartate (NMDA)receptor activation at CA1 and CA3 synapses, respec-tively (Malenka and Nicoll, 1999). Short-term synapticfacilitation induced by LFS was also shown to be syn-apse-specific at least in the CA3 area of the hippocam-pus. Indeed, this facilitation is generally stronger whenthe LFS protocol is applied to the mossy fibers than tothe association/commissural fibers (Salin et al., 1996).In addition, this LFS-induced short-term facilitation atthe synapses between mossy fibers and CA3 neuronsappears to involve the stimulation of presynaptic kai-nate receptors (Lauri et al., 2001; Schmitz et al., 2001)and to be regulated by presynaptic metabotropic gluta-mate receptors (Scanziani et al., 1997).

Taken together, these data support the hypothesis thatLFS-mediated changes in synaptic efficacy could beregion-specific in the hippocampal formation and thusthat LFS may not have the same consequences on synap-tic efficacy according to the hippocampal area where it isapplied. To examine the regional distribution of theseLFS-mediated alterations of synaptic transmission withinthe rat hippocampal formation accurately, we have useda multi-electrode array to record synaptic activity.Indeed, this technique permits multiple simultaneousrecordings at fixed distances from the stimulation.

MATERIALS AND METHODS

Drugs

D-(�)-2-amino-5-phosphonopentanoic acid (D-AP5),2,3-dihydro-6-nitro-7-sulfamoyl-benzo(f )quinoxaline(NBQX), 2-methyl-6-(phenylethynyl)pyridine (MPEP),(RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) and(S)-(þ)-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385) were from Tocris Cookson. Kynurenic acid, 1,4-

Laboratoire ‘‘Plasticite Cerebrale,’’ FRE 2693 CNRS, Universite Mont-pellier II, 34095 Montpellier cedex 05, FranceGrant sponsors: French Ministry of Higher Education, Centre Nationalpour la Recherche Scientifique (CNRS), and Region Languedoc-Roussillon.*Correspondence to: Michel Vignes; Laboratoire ‘‘Plasticite Cerebrale,’’FRE 2693 CNRS, Universite Montpellier II, 34095 Montpellier cedex 05,France. E-mail: [email protected] for publication 16 October 2005DOI 10.1002/hipo.20146Published online 21 November 2005 in Wiley InterScience (www.interscience.wiley.com).

HIPPOCAMPUS 16:345–360 (2006)

VVC 2005 WILEY-LISS, INC.

diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene(U0126), N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesul-fonamide (H-89), 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine(H-7), forskolin, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD098059), and picrotoxin were obtained from Sigma.

Hippocampal Slice Preparation

Experiments were carried out on hippocampal slices (350 lm)obtained from adult Sprague-Dawley rats (150–200 g) of bothsexes. All animal procedures were conducted in strict adherencewith the European Community Council Directive of 24 Novem-ber 1986 (86–609/EEC). After decapitation, brains were quicklydissected and placed in ice-cold buffer comprising the following:124 mM NaCl, 3.5 mM KCl, 25 mM NaHCO3, 1.25 mMNaH2PO4, 1 mM CaCl2, 2 mM MgSO4, and 10 mM Glucose(bubbled with O2/CO2:95/5%). Slices were then prepared witha vibratome (VT1000S Leica) and maintained at room tempera-ture for at least 1 h in the same buffer supplemented with1 mM CaCl2. This latter buffer was used for further recordings.

Electrophysiological Recordings WithMicroelectrode Arrays

For electrophysiological recordings, slices were transferred toa multi-electrode array (MEA-60 Multichannelsystems) com-prising 60 extracellular electrodes. The interelectrode distancewas 150 lm. Any individual electrode from the array could beused as recording or stimulatory electrode. A nylon mesh waspositioned above the slice to obtain a satisfactory electrical con-tact between the surface of the slice and the electrode array.Slices were continually superfused with the extracellularmedium described above (flow rate, 2 ml min�1) and main-tained at 378C. Picrotoxin (25 lM) was included in the per-fusate to eliminate GABAA-mediated synaptic transmission.Monopolar stimulation was achieved with an external stimula-tor (MC Stimulus, Multichannelsystems) by applying biphasiccurrent pulses to one electrode of the array. Stimulation inten-sity (20–200 lA) and duration (70–100 ls) were adapted toavoid multiphasic responses due to an excessive stimulation(Heuschkel et al., 2002). Field EPSPs could then be recordedby the remaining electrodes of the array at the same time. Testfrequency to evoke fEPSPs was 0.066 Hz. Slices that displayedepileptic-like activity were discarded. Field EPSPs were recorded

and analyzed (MC Rack, Multichannelsystems). The stimula-tion of the dendritic fields of hippocampal neurons resulted inthe occurrence of fEPSPs in the recording electrodes that wereblocked by non-NMDA receptor antagonist, NBQX (10 lM),and by tetrodotoxin (500 nM) (Fig. 1A, B). An individualexperiment (n) was performed on an individual rat.

Electrophysiological Recordings WithGlass Microelectrodes

Hippocampal slices were transferred to a submerged-stylerecording chamber positioned under an upright microscope(DMF, Leica) and superfused with the extracellular medium(flow rate, 2 ml min�1). Field EPSPs were recorded in the den-dritic field of CA1 hippocampal neurons with glass microelectr-odes (4–5 MX resistance) filled with extracellular mediumdescribed above. Field EPSPs were evoked by test frequencystimulation (0.066 Hz) of the Schaffer collateral–commissuralpathway using a bipolar electrode. In all experiments, the con-tribution of GABAA-mediated inhibitory synaptic potentialswas eliminated by including picrotoxin (25 lM) in the perfu-sate. Electrophysiological signals were amplified (Axopatch200B, Axon Instruments) and digitized (Digidata 1200A, AxonInstruments). Field EPSPs were further recorded and analyzedwith Dr. W. Anderson’s LTP software (Anderson and Colling-ridge, 2001). Experiments were carried out at 378C.

Measurement of cAMP Concentration

Cyclic AMP concentration was measured in CA1 microslicesprepared by carefully dissecting the area located in the immedi-ate vicinity of the stimulating electrode. These dissections wereperformed immediately after either LFS (1 Hz, 5 min) or teststimulation in the presence or in the absence of MPEP (10 lM).Microslices were sonicated in ice-cold HCl (0.1 N) to inactivatephosphodiesterases and centrifuged (15 min; 20,000 g; 48C).Supernatants were collected and stored at �808C for furthermeasurement of cAMP. Pellets were dissolved in NaOH (0.1 N)and stored at �208C for protein determination. The cAMP con-centration was determined using Correlate-EIA kit (Assays De-signs, USA), following the instructions of the manufacturer. Pro-tein concentration was measured using Bradford assay (Bio-Radprotein assay). The protein content per microslice was in therange of 6–12 lg.

FIGURE 1. Long lasting enhancement of synaptic transmissionby the application of LFS selectively induced in CA1 area. A, B:Sensitivity to TTX (500 nM) and NBQX (10 mM) of evoked syn-aptic potentials recorded in the CA1 area by stimulation of theSchaffer collateral/commissural fiber pathway. C–E: Effect of theapplication of LFS (1 Hz, 5 min) in the CA1 area. C: Schematicdiagram showing a hippocampal slice positioned on the MEA withthe delimited area of the CA1 area in which synaptic plasticitycould be observed. D: Pooled data obtained from six distinct ex-periments. Data presented are those obtained from the immedi-ately adjacent electrodes to the stimulating one, which can be anyof the delimited area. E: Synaptic enhancement obtained 20 minafter 1-Hz stimulation (5 min) respectively to the distance from

the stimulating electrode. To obtain this graph, signals originatingfrom the same row of electrodes, as shown in Figure 1C, wererecorded simultaneously. Field EPSPs recorded in adjacent (1,10)and distal (2,20) electrodes to the stimulating one (encircled) wereaveraged (n = 5) (*: significant from control, P < 0.05). F, G:Effect of the application of low frequency stimulation (1 Hz,5 min) in the CA3 area. F: On the schematic diagram, therecorded area has been delimited and the stimulating electrode(encircled) positioned in the mossy fiber pathway. G: Pooled dataobtained from five distinct experiments. Data presented are thoseobtained from the immediately adjacent electrodes to the stimulat-ing one. On graphs data are means (±SEM) and they are expressedas percentages of control fEPSP amplitude.

346 LANTE ET AL.

FIGURE 1

LOW FREQUENCY-INDUCED SLOW-ONSET POTENTIATION 347

MAP Kinase Phosphorylation Assay

To measure MAP kinase activation, CA1 minislices were pre-pared 20 min or 1 h after the application of a stimulation pro-tocol. Minislices were quickly frozen in liquid N2 and stored at–808C for further analysis. Slices receiving only test stimulationfrequency (i.e., 0.066 Hz) were used as control. MAP kinase(Erk1/2) phosphorylation was measured by Western blot, usinga phospho-specific antibody, antiphospho-p44/42 MAP kinase(Thr202/Tyr204) (Cell Signaling Technologies). Proteins wereextracted by sonication of minislices in 2% boiling SDS.Lysates were centrifuged for 10 min at 15,000 g at 108C. Pro-tein content in the supernatant was determined with a Lowry-based method (Bio-Rad DC-protein assay, Bio-Rad). In general,25–30 lg of protein was recovered from one minislice. Twentylg of proteins were electrophoresed on a 12% SDS-PAGE.Proteins were transferred onto nitrocellulose membrane usingFastblot half-dry blotting system (Biometra) in 25 mM Tris,192 mM glycine, and 10% methanol, at 200 mA for 24 min.Membrane was washed twice in phosphate buffered saline(PBS)–Tween, 0.1%, incubated for 1 h in PBS-Tween, 0.1%,bovine serum albumin (5%) and then overnight at 48C withphospho-Erk1/2 antibody (1/1,000) in the same buffer. Revela-tion was performed using peroxidase-conjugated secondary anti-body and enhanced chemiluminescence. Chemiluminescencesignals were recorded using Lumi-Imager F1 (Roche AppliedScience) and quantified with Lumi-Analyst software (RocheApplied Science). After stripping, membranes were reprobedwith antip 44/42 MAP kinase (1/1,000, Cell Signaling Tech-nologies) following the same protocol, so as to normalize thedata. Data are expressed as percentages of control values ob-tained in minislices stimulated at 0.066 Hz.

Statistical Analysis

For electrophysiology data, the statistical significance of the dif-ference between experimental and control data, obtained in sisterslices from the same animal, was assessed using unpaired Student’st-test (P < 0.05 considered significant and indicated by *).

RESULTS

The application of a LFS protocol (1 Hz, 5 min) with amicroelectrode array (MEA) in the CA1 and CA3 subfieldsresulted in a transient enhancement of fEPSP amplitude. On

average, 1-Hz stimulation increased fEPSP amplitude to130% 6 4% (n ¼ 6; Fig. 1C,D) and to 123% 6 3% (n ¼ 5;Fig. 1F,G) of control, in the CA1 and CA3 areas, respectively.When stimulating frequency was switched back to test value,i.e., 0.066 Hz, fEPSP amplitude returned to basal value in theCA3 area (101% 6 2% of control 10 min after the termina-tion of LFS; n ¼ 5; Fig. 1G). By contrast, in the stratum radia-tum of the CA1 area, the synaptic facilitation mediated by LFSwas followed by a long-lasting enhancement of synaptic trans-mission that was characterized by a slow onset. Indeed, a stablepotentiation of fEPSPs was obtained about 15 min after thetermination of LFS protocol (Fig. 1D). Therefore, data relativeto fEPSP potentiation were generally measured at least 15 minafter LFS application. On average, fEPSP amplitude repre-sented 159% 6 4% of control (n ¼ 29; minimal and maximalvalues obtained were 138 and 177%, respectively). Interestingly,this increase was observed only in a limited region of the CA1area located at a maximal distance of 150 lm underneath thecell body layer (see delimited area in Fig. 1C) and containingessentially the dendritic tree of CA1 pyramidal cells. In addi-tion, this potentiation was observed only in the two electrodesimmediately adjacent to, but no longer two electrodes awayfrom, the stimulating electrode, as shown by simultaneousrecordings performed in a row of electrodes (n ¼ 5; Fig. 1E).Such a potentiation was never observed anywhere else in thehippocampal formation. Consequently, further investigations onthis slow-onset LTP were restricted to recordings carried out inthe area delimited in the Figure 1C and only fEPSPs obtainedfrom the electrodes adjacent to the stimulating one were takeninto account.

We next examined whether this slow-onset potentiationcould be observed with a conventional electrophysiological set-up. For this, fEPSPs evoked with an extracellular bipolar elec-trode positioned in the Schaffer collateral–commissural fiberswere recorded with an extracellular glass microelectrode in thedendritic subfield of CA1 hippocampal neurons. Under theserecording conditions, the application of 1-Hz stimulation for5 min did not trigger synaptic plasticity, except when the stim-ulating electrode was positioned sufficiently close to the record-ing one (�200 lm). In this latter case, LFS induced a slow-onset LTP (n ¼ 6; Fig. 2A) with a time-course similar to thatobtained with an MEA (n ¼ 4; Fig. 2C). However, this long-lasting synaptic enhancement recorded with a conventional set-up was consistently associated with an increase in the fiber vol-ley amplitude (Fig. 2B). Indeed, the afferent fiber volley ampli-

FIGURE 2. Comparison between conventional glass microelec-trode- and multielectrode array-based set-up to obtain the slow-onset LTP. A, B: The slow-onset LTP is associated with changes inpresynaptic fiber volley amplitude when a conventional electrophy-siological recording set-up is used. A: Pooled data obtained fromsix distinct experiments. On graph data are means (±SEM) andthey are expressed as percentages of control fEPSP amplitude. Rep-resentative traces from one experiment are shown. They wereextracted at the times indicated (1, 2) on the graph. B: Plot of theaveraged fiber volley amplitude (n = 6) obtained from the pooledfEPSPs in (A). On graph data are means (±SEM) and they are

expressed as percentages of control fiber volley amplitude. C, D:The slow-onset LTP evidenced using a MEA is not associated to achange of presynaptic fiber volley amplitude. C: Pooled data ob-tained from four distinct experiments. On graph, data are means(±SEM) and they are expressed as percentages of control fEPSPamplitude. Representative traces from one experiment are shown.They were extracted at the times indicated (1, 2) on the graph. D:Plot of the averaged fiber volley amplitude (n = 4) obtained fromthe pooled fEPSPs in (C). On graph, data are means (±SEM) andthey are expressed as percentages of control fiber volley amplitude.

348 LANTE ET AL.

tude was 212% 6 18% of control after synaptic plasticity hastaken place, i.e., 15 min after 1-Hz stimulation (correspondingfEPSP amplitude 195% 6 25%; Fig. 2A). By contrast, using a

MEA, the afferent fiber volley amplitude was unaffected by theapplication of the LFS protocol. It was 100% 6 4% of control(Fig. 2D; n ¼ 4) 15 min after the termination of the LFS pro-

FIGURE 2


tocol (corresponding fEPSP amplitude 162% 6 22%; Fig.2C). Therefore the slow-onset LTP obtained with a conven-tional set-up appeared to result not only from synaptic modifi-cations but also from the recruitment of new afferent fibers. Bycontrast, the use of an MEA prevented the recruitment of newfibers by afferent stimulation and allowed to evidence more dis-crete synaptic modifications. Therefore, the slow-onset LTP wasfurther characterized using only the MEA.

The duration of LFS application required to trigger thisslow-onset form of LTP was investigated. Slow-onset LTP wasconsistently observed after a 3-min stimulation at 1 Hz (150%6 17% of control; n ¼ 4), while 1 min at 1 Hz was inefficient(Fig. 3A,B). Moreover, in some slices, the induction of theslow-onset LTP could still be obtained even though a transientquickly regulating enhancement of fEPSPs was observed upon1-Hz stimulation (Fig. 3C; 202% 6 7% of control, n ¼ 5).Therefore, the occurrence of the slow-onset LTP was independ-ent from the degree of short-term plasticity observed during1-Hz stimulation. Conversely, the duration of the 1-Hz stimu-lation was critical. Paired-pulse facilitation ratio (PPFR), ob-tained by applying two stimuli with a 50 ms interval was onaverage 1.73 6 0.13 (n ¼ 5), under test stimulation frequency(0.066 Hz; Fig. 3D). PPFR was significantly (P < 0.05)diminished to 1.33 6 0.08 during the application of LFS.However, once the slow-onset LTP was induced, PPFR re-turned to 1.75 6 0.15 (Fig. 3D), a value not significantlydifferent from control PPFR. This result, along to the invaria-bility of afferent fiber volley amplitude, indicates that the ex-pression locus of this form of synaptic plasticity is mainlypostsynaptic.

Remarkably, the application of a second LFS protocol afterthe induction of the slow-onset LTP did not further increasethe fEPSP potentiation induced by the first LFS protocol. Inthe same slices, the application of HFS (HFS; 100 Hz, 1 s)subsequently enhanced fEPSP potentiation (n ¼ 4) (Fig. 3E).By contrast, LFS could reverse LTP induced by HFS of CA1afferent pathways (n ¼ 4; Fig. 3F), as previously establishedusing conventional recording set-up (Barrionuevo et al., 1980;Huang et al., 2001).

It should be noticed that the LFS-induced slow-onset LTPwas only observed in adult rat hippocampal slices. Indeed, wefound that in young animal slices (12–15 days old) 1-Hz stim-ulation applied for 5 min produced LTD (n ¼ 5; Fig. 3G), as

established by others using conventional electrophysiologicalmicroelectrode recording (Kemp and Bashir, 2001). Taken to-gether, these data strengthen the validity and the specificity ofMEA recordings along with the physiological relevance of LFS-elicited slow-onset LTP that we observed.

Next, we have examined whether ionotropic glutamate recep-tor activation could be involved in the triggering of this slow-onset LTP. NMDA receptors are classically involved in mostactivity-dependent synaptic plasticity phenomena elicited in thehippocampal CA1 area, such as LTP (Bliss and Collingridge,1993; Malenka and Nicoll, 1999). However, in the presence ofthe NMDA receptor antagonist AP5 (50 lM), the LFS-in-duced long-lasting enhancement of synaptic transmission couldstill be observed (138% 6 4% of control; n ¼ 4; Fig. 4A). Inaddition, when LFS protocol was applied in the presence of thebroad-spectrum ionotropic glutamate receptor antagonist, ky-nurenic acid (10 mM) (Yeckel et al., 1999), a robust synapticenhancement was still observed after washing out this antago-nist (290% 6 70% of control; n ¼ 4) (Fig. 4B). We verifiedthat AP5 (50 lM) and kynurenic acid (10 mM) completelyblocked NMDA-receptor mediated fEPSPs isolated in Mg2þ-free medium and in the presence of the non-NMDA receptorantagonist, NBQX (5 lM) (Fig. 4C). Therefore, ionotropicglutamate receptors do not appear to be directly involved inthe acquisition of this form of plasticity.

Some forms of hippocampal CA1 plasticity rely on phospho-lipase C-linked Group I metabotropic glutamate (mGlu) recep-tor activation (Bashir et al., 1993; Conn and Pin, 1997),including mGlu5 receptor subtype (Lu et al., 1997). We havetested this possibility by using the selective mGlu5 receptorantagonist MPEP (10–100 lM; Gasparini et al., 1999). In thepresence of this antagonist (10 lM, n ¼ 3; Fig. 5A), theinduction of the slow-onset LTP was completely inhibited(98.5% 6 5% of control, 20 min after 1-Hz stimulation). Inthe presence of kynurenic acid (10 mM), MPEP still blockedthe induction of this LTP (data not shown). LY367385 (100 lM;n ¼ 4), a selective mGlu1a receptor antagonist (Mannaioni et al.,2001; Rae and Irving, 2004), failed to block the induction of theslow-onset LTP (164% 6 25% of control fEPSP amplitude30 min after 1-Hz stimulation; Fig. 5B). It is noteworthy thatwhen applied, LY367385 consistently induced a reversible depres-sion of fEPSPs (Fig. 5B). These data suggest that the slow-onsetLTP is mediated by mGlu5 receptors, but not by mGlu1 ones. To

FIGURE 3. Characterization of slow-onset LTP in CA1 areatriggered by LFS. A, B: LFS duration threshold to obtain synapticplasticity. A: Effect of the duration of low frequency stimulationon fEPSP. On the graph, data relative to 1- or 3-min LFS applica-tion are shown. In each case, data are averages of four distinctexperiments. B: Pooled data of experiments in which LFS wasapplied for 1, 3, or 5 min. Data are means (±SEM) of normalizedfEPSP amplitude measured 15 min after the termination of LFSprotocol. C: Pooled data of experiments in which slow-onset LTPwas still obtained even though only a transient enhancement offEPSPs was observed during the 1-Hz stimulation (n = 5). D:Pooled data of paired-pulse ratio (n = 5). Paired-pulse facilitationwas obtained by delivering two stimuli with a 50 ms interval.

Paired-pulse ratio was calculated by dividing the second peakamplitude by the first one. Data are means (±SEM). Representativetraces have been extracted at different times: 5 min before (a), dur-ing (b) and 25 min after (c) the application of LFS (1 Hz, 5 min).E: Pooled data of four distinct experiments in which LFS protocol(1 Hz, 5 min) was applied twice prior to HFS (100 Hz, 1 s). F:LTP erasure by LFS. LTP was induced by applying HFS and LFSwas further applied. Data are means (±SEM) of four distinctexperiments and have been normalized to control fEPSP ampli-tude. G: Induction of LTD in immature rat (12–15 day-old) hip-pocampal slices by the application of LFS protocol (1 Hz, 5 min).Data are means (±SEM) of five distinct experiments and have beennormalized to control fEPSP amplitude.

350 LANTE ET AL.

further verify this conclusion, AIDA, another mGlu1 receptorantagonist, was tested. At concentrations below 1 mM, AIDA israther selective for mGlu1a receptors (Moroni et al., 1997). AIDA

(300 lM) did not block the induction of this plasticity (141% 67% of control fEPSP amplitude 30 min after 1-Hz stimulation; n¼ 4; Fig. 5B). Taken together, these data strongly indicate that

FIGURE 3


FIGURE 4. LFS-induced synaptic potentiation does not in-volve ionotropic glutamate receptors. A: Effect of the NMDAreceptor antagonist, AP5 (50 mM) on LFS-induced synaptic plasti-city. AP5 was present throughout the experiment. On graph, aver-ages of four individual experiments (±SEM) are presented. B:Effect of the broad-spectrum non-NMDA receptor antagonist,kynurenic acid (10 mM), on LFS-mediated synaptic enhancement.Kynurenic acid was applied for 10 min prior to LFS stimulationto ensure a complete blockade of synaptic activity, and washed out

immediately after the termination of the stimulation protocol. Ongraph, data are averages of four distinct experiments (±SEM). C:Representative example illustrating the blockade of NMDA recep-tor-mediated fEPSPs by AP5 (50 mM) and kynurenic acid(10 mM). NMDA receptor-mediated fEPSPs were isolated in thepresence of the non-NMDA receptor antagonist, NBQX (5 mM),and by omitting Mg2+ from the external medium. Data are repre-sentative of three independent experiments.

352 LANTE ET AL.

FIGURE 5 (Overleaf.)


mGlu1a receptor activation is not required for the induction ofthis slow-onset LTP.

Next, we have examined whether Ca2þ release from intracel-lular stores could be involved in the induction of this slow-onset LTP. For this purpose, thapsigargin, which induces intra-cellular Ca2þ stores depletion, was tested. Thapsigargin (10 lM;n ¼ 3) completely inhibited the induction of the slow-onsetLTP (Fig. 5C).

Protein kinase A (PKA) activation appears essential for manyforms of hippocampal plasticity both at the level of inductionand maintenance. For instance, PKA is involved in the consoli-dation of CA1 LTP (Frey et al., 1993; reviewed by Nguyenand Woo, 2003). In this respect, inhibition of PKA activity byH-7 (30 lM) or with more selective inhibitor, H-89 (0.5 lM)completely blocked the occurrence of the slow-onset LTP.Indeed, fEPSP amplitude was 99% 6 2% of control (n ¼ 3)or 95% 6 5% of control (n ¼ 6; Fig. 6A) 15 min after LFSapplication in the presence of H-7 or H-89, respectively. Incontrol experiments performed in parallel on sister slices, LFSmediated an increase in fEPSP amplitude to 160% 6 15% ofcontrol (Fig. 6A).

Since PKA activation appears to be required for the induc-tion of the slow-onset LTP, we have next examined whether1-Hz stimulation could stimulate adenylyl cyclase, resulting inan increase in cAMP concentration. To this aim, cAMP wasmeasured directly in the stimulated area of the CA1 subfield.We observed that 1-Hz stimulation led to a significant increasein cAMP concentration (111 6 23 vs. 62.4 6 7.3 pmol/mgprot under test stimulation; n ¼ 5). This increase was inhibitedby 10 lM MPEP (Fig. 6B). Then, we have tested whether thedirect stimulation of adenylyl cyclase by the application of for-skolin (10 lM, 20 min), which strongly increased cAMP con-centration (1,384 6 545 pmol/mg prot; n ¼ 5) could alsotrigger a long-lasting enhancement of excitatory transmission.Actually, forskolin mediated a strong and enduring enhance-ment of fEPSP amplitude (262% 6 24% of control, measured35 min after forskolin wash out, n ¼ 4, Fig. 6C), which wasinhibited when H-89 (0.5 lM) was applied prior to forskolin(94% 6 10% of control, measured 35 min after forskolinwashout, n ¼ 4; Fig. 6C). PPFR was not significantly alteredfrom control once forskolin-induced synaptic enhancement wasobtained (PPFR of 1.37 6 0.13 in control vs. 1.42 6 0.17when forskolin-mediated synaptic plasticity has taken place;P ¼ 0.68, n ¼ 4). Thus forskolin induced a synaptic facilita-tion mainly via postsynaptic mechanisms in a way similar to

that induced by 1-Hz stimulation. The requirement of PKAactivation to trigger the slow-onset LTP was further substanti-ated by occlusion experiments between 1-Hz stimulation andforskolin. First, forskolin did not further enhance 1 Hz inducedsynaptic potentiation (Fig. 6D; n ¼ 4). Second, forskolin (10lM) occluded the slow-onset LTP mediated by 1-Hz stimula-tion (153% 6 3% of control measured 15 min after forskolinwashout, n ¼ 4; Fig. 6E). It should be noticed that in the pres-ence of forskolin, 1-Hz stimulation induced a strong inhibitionof synaptic transmission (50% 6 10% of control), whichreversed when the stimulation was switched back to test fre-quency (Fig. 6E).

Downstream the activation of PKA, MAP kinase pathwaycan be stimulated (Roberson et al., 1999; reviewed by Sweatt,2001). To some extent, this pathway appears to be required forthe consolidation of synaptic plasticity in several experimentalmodels (Kanterewicz et al., 2000; Sweatt, 2001; Lynch, 2004).We have examined the putative involvement of MAP kinasepathway in the LFS-induced slow-onset LTP. The applicationof the MEK inhibitor U0126 (10 lM) did not block the long-lasting synaptic enhancement obtained with LFS (153% 613% of control vs. 164% 6 22% obtained in the absence ofU0126 in experiments performed in parallel on sister slices;n ¼ 4; Fig. 7A). Similar data were obtained with anotherMEK inhibitor, PD098059. Indeed, in the presence ofPD098059 (50 lM; applied 15 min prior to stimulation), 1-Hzstimulation could still induce a slow-onset LTP (152% 6 10%of control fEPSP amplitude 20 min after the termination ofLFS, n ¼ 3). A previous report (Kanterewicz et al., 2000) indi-cated that the blockade of synaptic plasticity by MEK inhibitionin CA1 area was observed only 20 min after MEK inhibitorwashout, high frequency stimulation being delivered in the pres-ence of the inhibitor. We did not observe any inhibition even60 min after MEK inhibitor washout (135% 6 3% of controlamplitude, n ¼ 3; Fig. 7B). In addition, a long lasting forsko-lin-mediated synaptic enhancement (296% 6 55% of control; n¼ 3) was still observed in the presence of U0126 (Fig. 7C).

In parallel, ERK1/2 phosphorylation status was investigatedin response to LFS and forskolin in CA1 minislices collectedafter electrophysiological recordings (Fig. 8A,B). It appearedthat ERK1/2 phosphorylation was significantly enhanced by 1-Hz stimulation (204% 6 38% of control; n ¼ 4). PKA block-ade with H-89 had no effect on this phosphorylation, and evenenhanced it, although not significantly (279% 6 27%; n ¼ 4).We verified that U0126 completely inhibited ERK1/2 phos-

FIGURE 5. Involvement of mGlu5 receptor and intracellularcalcium mobilization in LFS-induced synaptic potentiation. A: Ef-fect of mGlu5 receptor blockade with MPEP (10 mM) on LFS-induced synaptic enhancement. MPEP was applied for 5 min priorto LFS stimulation, and washed out 5 min after the end of stimu-lation protocol. Data are averages of five distinct experiments(±SEM). B: Lack of effect of mGlu1a receptor blockade with AIDA(300 mM; filled circles) or LY367385 (100 mM; open circles),respectively, on LFS-induced synaptic potentiation. AIDA orLY367385 were applied for 10 min prior to LFS stimulation, andwashed out 5 min after the end of stimulation protocol. Data are

averages of five distinct experiments (±SEM). On the right, sampletraces of an experiment performed in the presence of AIDA andextracted at the indicated time points are shown. C: Dependenceof the slow-onset LTP on intracellular Ca2+ store mobilization.Thapsigargin (10 mM) was applied 15 min prior to 1-Hz stimula-tion. Data are means (±SEM) of normalized fEPSP amplitude ofthree distinct experiments. D: Summary graph depicting the effectof various drug treatments on LFS-induced synaptic plasticity. Onthe graph, normalized fEPSP amplitudes (±SEM) measured 15 minafter the termination of LFS protocol are shown.

354 LANTE ET AL.

phorylation to a level even lower than that in control slicestaken 20 min (15% 6 5% of control; n ¼ 4) or 60 min (10% 62% of control; n ¼ 3) after the termination of the LFS proto-col. Forskolin (10 lM) also dramatically enhanced ERK1/2

phosphorylation (259% 6 38% of control) in an H-89-insensi-tive manner (293% 6 81% of control). In the presence ofU0126, forskolin-induced ERK1/2 phosphorylation was com-pletely blocked (23% 6 8% of control; n ¼ 3). Interestingly,

FIGURE 6. PKA activation dependency of LFS-mediated syn-aptic increase. A: Pooled data of the action of the PKA inhibitorH-89. H-89 (0.5 mM) was included in the perfusate for 10 minprior to LFS stimulation, and washed out 5 min after the termina-tion of the stimulation protocol. Control experiments (n = 6, opencircles) and those including H-89 (n = 6, filled circles) wereobtained from sister slices. Data are means (±SEM) of normalizedfEPSP amplitude. B: Concentrations of cAMP measured after test(0.066 Hz, Cont) and LFS (1 Hz, 5 min), either in the absence orin the presence of 10 mM MPEP. Data, expressed as pmol ofcAMP per mg of protein, are means (±SEM) of five independent

experiments. For each sample, cAMP dosage was performed intriplicate by EIA, as described in the ‘‘Materials and Methods’’ sec-tion. C: Pooled data of the synaptic enhancement obtained withforskolin (10 mM, 20 min) in the CA1 area either in the presence(open circles) or in the absence of the PKA inhibitor H-89 (0.5mM; filled circles) (n = 4, each condition). D, E: Pooled data ofocclusion experiments between forskolin (10 mM) and 1-Hz stimu-lation (n = 4 in each case). In (D), 1Hz stimulation preceded for-skolin application. In (E), 1-Hz stimulation was applied in thepresence of forskolin when a stable synaptic enhancement wasobserved. Data are means (±SEM) of normalized fEPSP amplitude.


FIGURE 7

356 LANTE ET AL.

in the presence of AIDA, LFS protocol was still able to stimu-late ERK1/2 phosphorylation (173% 6 30% of control; n ¼3). By contrast, this phosphorylation was completely inhibitedby MPEP (24% 6 1% of control; n ¼ 3). Remarkably, in thepresence of MPEP, ERK1/2 phosphorylation level was similarto that obtained in naıve slices, i.e., slices neither treated withpicrotoxin, nor electrically stimulated at 378C. This suggestedthat basal ERK1/2 phosphorylation in control slices, i.e., stimu-lated at 0.066 Hz in the presence of picrotoxin at 378C, partlyaccounted for mGlu5 receptor activation.

DISCUSSION

The use of an MEA to record synaptic activity and to stimu-late synaptic pathways enabled us to demonstrate that theapplication of a low frequency (1 Hz) of stimulation for 3–5 min, which is generally considered as innocuous to synapticactivity, triggers a long-lasting enhancement of synaptic trans-mission. This synaptic enhancement is characterized by a slow-onset. Previous reports have indicated that repetitive stimula-tions of mossy fibers afferent to CA3 neurons lead to a transientenhancement of synaptic transmission during the stimulation(Salin et al., 1996; Scanziani et al., 1997; Vignes et al., 1998).We find here that LFS elicits comparable transient increasesin synaptic transmission in both CA1 and CA3 areas during1-Hz stimulation. However, the slow-onset LTP occurs specifi-cally in the CA1, but not in the CA3, area. In addition, it isobserved in a very limited area located in the vicinity of thecell body layer. This indicates that LFS leads to the long-last-ing potentiation of synapses located close to the cell body ofCA1 neurons. Although synaptic potentials can be recorded ina row of electrodes when a stimulus is applied at test frequency(0.066 Hz) to one electrode of the row, plasticity is restrictedto the two electrodes immediately adjacent to the stimulatingone. Therefore, the stimulation of synaptic pathways via theMEA leads to the potentiation of only a small number of syn-apses surrounding the stimulating electrode.

Although paired-pulse ratio is decreased during LFS applica-tion, suggesting an increase in glutamate release, it is not differ-ent from control after the induction of synaptic plasticity. Thissuggests that the locus of expression of this slow-onset LTPobserved here is probably mainly postsynaptic. In addition,fiber volley amplitude does not change when this synaptic plas-ticity has taken place. This precludes any recruitment of newfibers to explain the increased synaptic activity. By contrast,when a conventional electrophysiological set-up is used, the

slow-onset LTP observed after 1-Hz stimulation is essentiallydue to the recruitment of new fibers as evidenced by an in-crease in fiber volley amplitude. Interestingly, Herron and Mal-enka, (1994) have obtained a similar form of synaptic plasticityby applying 1 Hz for 5 min with a conventional set-up, butsolely in the presence of the phosphatase inhibitor calyculin A.Under these conditions, the slow-onset LTP has been attributedto presynaptic changes, since it was associated with paired-pulseratio decrease and fiber volley change (Herron and Malenka,1994).

The slow-onset LTP, that we evidence here, is independentof ionotropic (NMDA and non-NMDA) glutamate receptorstimulation as shown by pharmacological manipulations. Thisis further strengthened by the fact that NMDA-dependent LTPhas additive effect with 1 Hz induced potentiation. Remark-ably, an enduring form of synaptic plasticity independent ofNMDA receptor activation and induced by low frequency(1 Hz, 15 min) has recently been described in the amygdala(Li et al., 1998). This enhancement seems to require GluR5kainate receptor activation (Li et al., 2001). In our hands, thedirect activation of GluR5 kainate receptors with the selectiveagonist, ATPA (1 lM), results only in a synaptic depression, aspreviously demonstrated (Vignes et al., 1998), but does nottrigger a slow-onset LTP in CA1 hippocampal area (data notshown). In addition, the blockade of ionotropic glutamatereceptors with kynurenic acid does not inhibit LFS-inducedsynaptic enhancement. On the contrary, this blockade seemsrather to have a facilitatory action on the slow-onset LTP. Thissuggests that non-NMDA receptor activation during LFS appli-cation may exert a negative modulatory effect on the expressionof LFS-induced plasticity. In this respect, presynaptic kainatereceptors may negatively regulate excitatory synaptic transmis-sion in the CA1 area of the hippocampus (Chittajallu et al.,1996). Therefore, these receptors could be activated duringLFS, exerting a negative feedback on glutamate synaptic release.Although not required for the induction, our data do not pre-clude any involvement of ionotropic glutamate receptors in thelate expression of this slow-onset LTP.

By contrast, PLC-linked mGlu5 receptor activation is neces-sary to trigger the slow-onset LTP, as evidenced by the inhibi-tory effect of the mGlu5 receptor antagonist, MPEP. Thesereceptors are found essentially at postsynaptic sites on pyrami-dal hippocampal cells (Romano et al., 1995; Lujan et al.,1996), while mGlu1 receptors are more abundantly located oninterneurons (Ferraguti et al., 2004). This distinct locationcould explain why mGlu1a receptor blockade with either AIDAor LY367385 does not inhibit the slow-onset LTP. The prefer-

FIGURE 7. LFS-induced synaptic enhancement occurs inde-pendently of ERK activation. A: Lack of effect of MEK inhibitionon LFS-mediated synaptic plasticity. The MEK inhibitor U0126(10 mM) was applied 15 min before LFS stimulation and washedout 5 min after the termination of the protocol. Control experi-ments (n = 4, open circles) and those including U0126 (n = 4,filled circles) were performed on sister slices. B: Lack of effect of

U0126 (10 mM) over a longer time scale (n = 3). C: Lack of effectof U0126 on forskolin-induced synaptic enhancement. For theseexperiments, U0126 was applied 15 min before exposure to forsko-lin (10 mM, 20 min) and washed out at the same time as that offorskolin. On graphs, data are means (±SEM) of normalizedfEPSP amplitude.


ential location of mGlu5 receptors at postsynaptic sites in theCA1 area further supports a postsynaptic nature of this slow-onset LTP. Interestingly, the slow-onset LTP that we describehere shares a lot of features with mGlu receptor-induced slow-onset LTP obtained by the application of the mGlu receptoragonist, 1S,3R-ACPD, in CA1 area (Bortolotto et al., 1994;Bortolotto and Collingridge, 1995). Indeed, mGlu receptor-induced LTP is independent of NMDA receptor activation,requires intracellular Ca2þ elevation, and is not associated witha change in fiber volley (Bortolotto and Collingridge, 1995).

From our data, one can suggest that LFS of CA1 neuron affer-ent fibers reproduces the slow-onset LTP obtained by agonist-mediated mGlu receptor stimulation. Therefore, this protocolleads to the release of an amount of glutamate sufficient toactivate mGlu5 receptors synaptically and to trigger synapticplasticity. In this respect, at least 3 min of 1-Hz stimulation arerequired to trigger the slow-onset LTP.

This plasticity requires intracellular Ca2þ increase probablymediated by intracellular Ca2þ release from intracellular stores.Ca2þ may activate adenylyl cyclase, thus increasing cAMP leveland therefore activating PKA. In this respect, we find that LFSleads to an increase in cAMP content in CA1 neurons proximalto the stimulation point in a mGlu5 receptor-dependent fash-ion. Interestingly, HFS of Schaffer collateral inputs, which trig-gers NMDA receptor-dependent LTP in CA1 area, is also asso-ciated with an elevation of cAMP concentration (Chetkovichet al., 1991). Therefore, an increase in cAMP content could bea common step for these two forms of plasticity. In this view,PKA activation is largely involved in a broad spectrum of hip-pocampal synaptic plasticity phenomena. More precisely, it haspreviously been demonstrated that PKA is involved in the latephase of hippocampal LTP (Frey et al., 1993; Nguyen andWoo, 2003). However, in the CA1 area, PKA inhibition doesnot appear to block LTP induction completely (Otmakhovaet al., 2000). We find here that PKA activation is absolutelyrequired for the induction of the slow-onset LTP. Previouswork indicates that a PKA-dependent form of synaptic plasti-city could be induced by the application of 5 Hz for 30 s inthe CA1 area of the mouse hippocampus (Thomas et al.,1996). Therefore, by comparison with high-frequency inducedLTP, which involves a large number of kinases, including CaM-KII, PKA activation appears to be an obligatory step in theexpression of low-frequency induced synaptic plasticity. This isconfirmed by the fact that direct activation of adenylyl cyclasewith forskolin and LFS occlude each other effect on synapticplasticity.

Although PKA activation is shown to trigger ERK phos-phorylation in several models, MAP kinase pathway does notappear to be necessary either to induce or to consolidate thisslow-onset LTP. Indeed, the blockade of ERK phosphorylationwith the MEK inhibitor U0126 prevents neither LFS-, norforskolin-induced synaptic plasticity. ERK phosphorylationhas been implicated in hippocampal synaptic plasticity(Sweatt, 2001), especially in NMDA-receptor dependentforms of LTP. On the other hand, it has recently beenreported that in the CA1 area of the rat hippocampus, anNMDA receptor independent form of LTP was dependent onERK phosphorylation (Kantarewicz et al., 2000). However, itappears that in the CA1 area of the mouse hippocampus, LTPinduction is largely insensitive to MEK inhibitors (Opazoet al., 2003). From our data, although LFS and forskolin pro-mote ERK phosphorylation, this phosphorylation is notrequired for the expression of this plasticity and is insensitiveto PKA inhibition. This suggests that PKA activation inducedby LFS is not required for ERK phosphorylation. In thisrespect, it has been reported that an enhancement of cAMP

FIGURE 8. ERK1/2 phosphorylation by LFS is not requiredfor LFS-induced synaptic plasticity in CA1 area. A: Representativephospho-ERK Western blots from control, 1 Hz-treated and For-skolin (FSK, 10 mM)-treated CA1 minislices. Controls correspondto slices stimulated at test frequency (0.066 Hz) throughout therecording. Data obtained from slices receiving 1-Hz stimulation inthe presence of PKA inhibitor (H-89), MEK inhibitor (U0126),mGlu1 and mGlu5 receptor antagonists (AIDA and MPEP, respec-tively), and slices treated with forskolin (FSK) in the presence ofPKA (H-89) or MEK (U0126) inhibitors are also shown. B:Pooled data of ERK1 + ERK2 phosphorylation. Phospho-ERK1/2immunoreactivity has been normalized to total ERK1/2 immunor-eactivity and then expressed as percentages of control obtained intest frequency stimulated slices. Data are means (±SEM) of at leastthree independent determinations.

358 LANTE ET AL.

concentration is sufficient to promote ERK phosphorylationindependent of PKA activation (Ding et al., 2002; Charleset al., 2003). More precisely, it is suggested that ERK phos-phorylation in response to an elevation of cAMP couldinvolve Rap-1 pathway in PC-12 cells independent of PKAstimulation (Charles et al., 2003). The activation of such apathway in CA1 hippocampal neurons could thus explain whyERK phosphorylation can be obtained in a PKA-independentway, following the induction of the slow-onset LTP.

REFERENCES

Anderson WW, Collingridge GL. 2001. The LTP Program: a dataacquisition program for on-line analysis of long-term potentiationand other synaptic events. J Neurosci Methods 108:71–83.

Barrionuevo G, Schottler F, Lynch G. 1980. The effects of repetitivelow frequency stimulation on control and ‘‘potentiated’’ synapticresponses in the hippocampus. Life Sci 27:2385–2391.

Bashir ZI, Bortolotto ZA, Davies CH, Berretta N, Irving AJ, Seal A,Henley JM, Jane DE, Watkins JC, Collingridge GL. 1993. Induc-tion of LTP in the hippocampus needs synaptic activation of gluta-mate metabotropic receptors. Nature 363:347–350.

Bliss TVP, Collingridge GL. 1993. A synaptic model of memory long-term potentiation in the hippocampus. Nature 361:31–39.

Bortolotto ZA, Collingridge GL. 1995. On the mechanism of long-term potentiation induced by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) in rat hippocampal slices. Neuropharma-cology 34:1003–1014.

Bortolotto ZA, Bashir ZI, Davies CH, Collingridge GL. 1994. Amolecular switch activated by metabotropic glutamate receptorsregulates induction of long-term potentiation. Nature 368:740–743.

Charles MP, Adamski D, Kholler B, Pelletier L, Berger F, Wion D.2003. Induction of neurite outgrowth in PC12 cells by the bacterialnucleoside N6-methyldeoxyadenosine is mediated through adenosineA2a receptors and via cAMP and MAPK signaling pathways. Bio-chem Biophys Res Commun 304:795–800.

Chetkovich DM, Gray R, Johnston D, Sweatt JD. 1991. N-methyl-D-aspartate receptor activation increases cAMP levels and voltage-gated Ca2þ channel activity in area CA1 of hippocampus. ProcNatl Acad Sci USA 88:6467–6471.

Chittajallu R, Vignes M, Dev KK, Barnes JM, Collingridge GL, Hen-ley JM. 1996. Regulation of glutamate release by presynaptic kai-nate receptors in the hippocampus. Nature 379:78–81.

Conn PJ, Pin JP. 1997. Pharmacology and functions of metabotropicglutamate receptors. Annu Rev Pharmacol Toxicol 37:205–237.

Ding WQ, Dong M, Ninova D, Holicky EL, Stegall MD, Miller LJ.2002. Forskolin suppresses insulin gene transcription in islet b-cellsthrough a protein kinase A-independent pathway. Cell Signal 15:27–35.

Ferraguti F, Cobden P, Pollard M, Cope D, Shigemoto R, Watanabe M,Somogyi P. 2004. Immunolocalization of metobotropic glutamatereceptor 1a (mGluR1a) in distinct classes of interneuron in theCA1 region of the rat hippocampus. Hippocampus 14:193–215.

Frey U, Huang YY, Kandel ER. 1993. Effects of cAMP simulate a latestage of LTP in hippocampal CA1 neurons. Science 260:1661–1664.

Gasparini F, Lingenhohl K, Stoehr N, Flor PJ, Heinrich M, VranesicI, Biollaz M, Allgeier H, Heckendorn R, Urwyler S, Varney MA,Johnson EC, Hess S, Rao SP, Sacaan AI, Santori EM, Velicelebi G,Kuhn R. 1999. 2-Methyl-6-(phenylethynyl)-pyridine (MPEP), apotent, selective and systemically active mGlu5 receptor antagonist.Neuropharmacology 38:1493–1503.

Herron CE, Malenka RC. 1994. Activity-dependent enhancement ofsynaptic transmission in hippocampal slices treated with the phos-phatase inhibitor calyculin A. J Neurosci 14:6013–6020.

Heuschkel MO, Fejtl M, Raggenbass M, Bertrand D, Renaud P. 2002. Athree-dimensional multi-electrode array for multi-site stimulation andrecording in acute brain slices. J Neurosci Methods 114:135–148.

Huang CC, Liang YC, Hsu KS. 2001. Characterization of the mecha-nism underlying the reversal of long term potentiation by low fre-quency stimulation at hippocampal CA1 synapses. J Biol Chem 276:48108–48117.

Kanterewicz BI, Urban NN, McMahon DBT, Norman ED, Giffen LJ,Favata MF, Scherle PA, Trzaskops JM, Barrionuevo G, Klann E.2000. The extracellular signal-regulated kinase cascade is requiredfor NMDA receptor-independent LTP in area CA1 but not areaCA3 of the hippocampus. J Neurosci 20:3057–3066.

Kemp N, Bashir ZI. 2001. Long-term depression: a cascade of induc-tion and expression mechanisms. Prog Neurobiol 65:339–365.

Lauri SE, Bortolotto ZA, Bleakman D, Ornstein PL, Lodge D,Isaac JTR, Collingridge GL. 2001. A critical role of a facilita-tory presynaptic kainate receptor in mossy fiber LTP. Neuron32:697–709.

Li H, Weiss SRB, Chuang D-M, Post RM, Rogawski MA. 1998.Bidirectional synaptic plasticity in the rat basolateral amygdala:characterization of an activity-dependent switch sensitive to thepresynaptic metabotropic glutamate receptor antagonist 2S-a-ethyl-glutamic acid. J Neurosci 18:1662–1670.

Li H, Chen A, Xing G, Wei ML, Rogawski MA. 2001. Kainate recep-tor–mediated heterosynaptic facilitation in the amygdala. Nat Neu-rosci 4:612–620.

Lu Y, Jia Z, Janus C, Henderson JT, Gerlai R, Wojtowicz JM, RoderJC. 1997. Mice lacking metabotropic glutamate receptor 5 showimpaired learning and reduced CA1 long-term potentiation (LTP)but normal CA3 LTP. J Neurosci 17:5196–5205.

Lynch MA. 2004. Long term potentiation and memory. Physiol Rev84:87–136.

Lujan R, Nusser Z, Roberts JD, Shigemoto R, Somogyi P. 1996. Peri-synaptic location of metabotropic glutamate receptors mGluR1 andmGluR5 on dendrites and dendritic spines in the rat hippocampus.Eur J Neurosci 8:1488–1500.

Malenka RC, Nicoll RA. 1999. Long-term potentiation—a decade ofprogress? Science 285:1870–1874.

Mannaioni G, Marino MJ, Valenti O, Traynelis SF, Conn PJ. 2001.Metabotropic glutamate receptors 1 and 5 differentially regulateCA1 pyramidal cell function. J Neurosci 21:5925–5934.

Moroni F, Lombardi G, Thomsen C, Leonardi P, Atucci S, PeruginelliF, Albani Torregrossa S, Pellegrini-Giampietro DE, Luneia R, Pelli-ciari R. 1997. Pharmacological characterization of 1-aminoindan-1,5-dicarboxylic acid, a potent m GluR1 antagonist. J PharmacolExp Ther 281:721–729.

Nguyen PV, Woo NH. 2003. Regulation of hippocampal synapticplasticity by cyclic AMP-dependent protein kinases. Prog Neuro-biol 71:401–437.

Nicoll RA, Malenka RC. 1995. Contrasting properties of two formsof long-term potentiation in the hippocampus. Nature 377:115–118.

Opazo P, Watabe AM, Grant SGN, O’Dell JT. 2003. Phosphatidylino-sitol 3-Kinase regulates the induction of long-term potentiationthrough extracellular signal related kinase-independent mechanisms.J Neurosci 23:3679–3688.

Otmakhova NA, Otmakhov N, Mortenson LH, Lisman JE. 2000.Inhibition of the cAMP pathway decreases early long-term poten-tiation at CA1 hippocampal synapses. J Neurosci 20:4446–4451.

Rae MG, Irving AJ. 2004. Both mGluR1 and mGluR5 mediate Ca2þ

release and inward currents in hippocampal CA1 pyramidal neu-rons. Neuropharmacology 46:1057–1069.

Roberson ED, English JD, Adams JP, Selcher JC, Kondratick C,Sweatt JD. 1999. The mitogen-activated protein kinase couples


PKA to cAMP response element binding protein phosphorylationin area CA1 of hippocampus. J Neurosci 19:4337–4348.

Romano C, Sesma MA, McDonald CT, O’Malley K, Van den Pol AN,Olney JW. 1995. Distribution of metabotropic glutamate receptormGluR5 immunoreactivity in rat brain. J Comp Neurol 355:455–469.

Salin PA, Scanziani M, Malenka RC, Nicoll RA. 1996. Distinct short-term plasticity at two excitatory synapses in the hippocampus. ProcNatl Acad Sci USA 93:13304–13309.

Scanziani M, Salin PA, Vogt KE, Malenka RC, Nicoll RA. 1997. Use-dependent increases in glutamate concentration activate presynapticmetabotropic glutamate receptors. Nature 385:630–634.

Schmitz D, Mellor J, Nicoll RA. 2001. Presynaptic kainate receptormediation of frequency facilitation at hippocampal mossy fiber syn-apses. Science 291:1972–1976.

Sweatt JD. 2001. The neuronal MAP kinase cascade: a biochemicalsignal integration system subserving synaptic plasticity and mem-ory. J Neurochem 76:1–10.

Thomas MJ, Moody TD, Makhinson M, O’Dell TJ. 1996. Activity-dependant b-adrenergic modulation of low frequency stimulationinduced LTP in the hippocampal CA1 region. Neuron 17:475–482.

Vignes M, Clarke VRJ, Parry MJ, Bleakman D, Lodge D, OrnsteinPL, Collingridge GL. 1998. The GluR5 subtype of kainate recep-tor regulates excitatory synaptic transmission in areas CA1 andCA3 of the rat hippocampus. Neuropharmacology 37:1269–1277.

Yeckel MF, Kapur A, Johnston D. 1999. Multiple forms of LTP inhippocampal CA3 neurons use a common postsynaptic mechanism.Nat Neurosci 2:625–633.

360 LANTE ET AL.

low-frequency stimulation induces a new form of ltp, metabotropic glutamate (mglu5) receptor- and...

Documents