Analysis of the Presynaptic Signaling MechanismsUnderlying the Inhibition of LTP in Rat Dentate Gyrusby the Tyrosine Kinase Inhibitor, Genistein

M. Casey, C. Maguire, A. Kelly, M.A. Gooney,and M.A. Lynch*

Physiology Department, Trinity College, Dublin, Ireland

ABSTRACT: A great deal of recent evidence points to a role for tyrosinekinase in expression of LTP. Data have been presented that are consistentwith the idea that tyrosine phosphorylation of proteins occurs in both thepresynaptic and postsynaptic areas. In this study, we set out to investigatethe role that tyrosine kinase might play presynaptically to modulaterelease of glutamate in an effort to understand the mechanism underlyingthe persistent increase in release that accompanies LTP in perforantpath–granule cell synapses. We report that LTP was associated withincreased calcium influx and glutamate release. LTP was also associatedwith an increase in phosphorylation of the �-subunit of calcium channelsand ERK in synaptosomes prepared from dentate gyrus, and these effectswere inhibited when LTP was blocked by the tyrosine kinase inhibitor,genistein. LTP was accompanied by increased protein synthesis and in-creased phosphorylation of CREB in entorhinal cortex, effects that werealso blocked by genistein. We conclude that tetanic stimulation leads toenhanced tyrosine phosphorylation of certain presynaptically located pro-teins that modulate glutamate release and contribute to expression of LTP.Hippocampus 2002;12:377–385. © 2002 Wiley-Liss, Inc.

KEY WORDS: glutamate release; calcium influx; ERK; CREB; calciumchannel �-subunit

INTRODUCTION

Among the changes that accompany long-term potentiation in dentategyrus is an increase in glutamate release that is inhibited when LTP isblocked, for example, by the NMDA receptor inhibitor, AP5 (Errington etal., 1987), the Trk inhibitor, tyrphostin AG879 (Maguire et al., 1999), theERK inhibitor, PD 98059 (McGahon et al., 1999), or when LTP is im-paired, for example, in aged rats (Murray and Lynch, 1998a,b). Thus, thereis a tight coupling between LTP and increased glutamate release pointing topresynaptic involvement in LTP in perforant path–granule cell synapses.

Several proteins kinases contribute to expression of LTP (see Bliss andCollingridge, 1993), and there is now convincing evidence that tyrosinekinase also plays a role. Consistent with this is the finding that tyrosine

kinase inhibitors block LTP (O’Dell et al., 1991; Abe andSaito, 1993; McGahon and Lynch, 1998; Lu et al., 1998;Maguire et al., 1999; Huang and Hsu, 1999), while LTPis accompanied by increased tyrosine phosphorylation ofsynaptophysin (Mullany and Lynch, 1998), PLC� (Mc-Gahon and Lynch, 1998) and the 2B subunit of theNMDA receptor (Rosenblum et al., 1996; Rostas et al.,1996). A role for at least two nonreceptor tyrosine ki-nases—fyn (Grant et al., 1992; Kojima et al., 1997) andsrc (Lu et al., 1998; Huang and Hsu, 1999)—has beendocumented in LTP, while receptor tyrosine kinases havealso been shown to be involved (Maguire et al., 1999;Kang and Schuman, 1995; Messaoudi et al., 1998).

An essential role for protein synthesis in maintainingthe long-lasting components of LTP has been identifiedby several laboratories (Charriaut-Marlangue et al.,1988; Otani et al., 1989; Fazeli et al., 1993; Mullany andLynch, 1997), but the trigger leading to the appropriatechanges remains to be identified. One transcription fac-tor, cAMP response element binding protein (CREB), isa candidate trigger, as it appears to mediate in the trans-duction of neuronal stimulation into gene expression(Ginty, 1997). Thus morphological changes in the spine(Murphy and Segal, 1997), late-phase LTP (Nguyen andKandel., 1996) and BDNF-induced transcription (Fink-beiner et al., 1997), all of which require protein synthesis,appear to rely on increased CREB phosphorylation.CREB phosphorylation also plays a role in LTP, perhapsas a consequence of increased activation of the mitogen-activated protein kinase, ERK (English and Sweatt,1997; McGahon and Lynch, 1998; Maguire et al., 1999;Davis et al., 2000) as shown directly (e.g. Impey et al.,1996; Davies et al., 2000) and by the observation thatLTP is impaired in CREB mutant mice (Bourtchuladzeet al., 1994).

In the present study, we focused on changes that occurpresynaptically after tetanization following treatmentwith saline or the tyrosine kinase inhibitor, genistein. Wereport that genistein inhibited expression of LTP in den-tate gyrus, as well as the LTP-associated increases in glu-tamate release and calcium influx. Our findings indi-cate that LTP was associated with increasedphosphorylation of ERK and the �-subunit of calcium

Grant sponsor: Health Research Board (Ireland); Grant sponsor: Forbairt(Ireland).*Correspondence to: M.A. Lynch, Physiology Department, Trinity College,Dublin 2, Ireland. E-mail: lynchma@tcd.ieAccepted for publication 24 October 2001DOI 10.1002/hipo.10036Published online 00 Month 2001 in Wiley InterScience (www.interscience.wiley.com).

HIPPOCAMPUS 12:377–385 (2002)

© 2002 WILEY-LISS, INC.

channels in synaptosomes prepared from dentate gyrus, both ofwhich are likely to enhance glutamate release, and increasedphosphorylation of CREB and protein synthesis in entorhinalcortex.

EXPERIMENTAL PROCEDURES

Animals

Male Wistar rats (250–350 g) were used in these experiments;rats were obtained from the BioResources Unit, Trinity CollegeDublin, where they were maintained under veterinary supervisionat an ambient temperature of 22–23°C, under a 12-h light sched-ule. The minimum number of rats was used compatible with sta-tistical analysis and every effort was made to minimize discomfort.All experimentation was conducted as required by local regulationsand conformed to Department of Health and Children (Ireland)and international guidelines on the ethical use of animals.

Induction of LTP In Vivo

Rats were anesthetized by intraperitoneal injection of urethane(1.5 g/kg i.p); LTP was induced as described previously (McGahonand Lynch, 1998). Briefly, a bipolar stimulating electrode wasplaced in the perforant path (4.4 mm lateral to lambda) and aunipolar recording electrode in the dorsal cell body region of thedentate gyrus (2.5 mm lateral and 3.9 mm posterior to the breg-ma). Test shocks were given at 30-s intervals for 10 min before, and40 min after, tetanic stimulation (3 trains of stimuli delivered at30-s intervals; 250 Hz for 200 ms). In some experiments, genistein(250 �M; 5 �l; Calbiochem, UK) or saline (5 �l) was injectedintracerebroventricularly (0.4 mm posterior to Bregma; 0.2 mmlateral to midline; 3.5-mm depth) 30 min before recording com-menced and the experiment proceeded as described. In a separateseries of experiments, the effect of delivering the same total numberof stimuli without the high-frequency train was assessed; in thiscase stimuli were delivered at a rate of 1 stimulus/12 s. At the endof the recording period, rats were killed by cervical dislocation,cross-chopped slices (350 �m) were prepared from ipsilateral andcontralateral dentate gyri and ipsilateral and contralateral entorhi-nal cortices. These samples were separately frozen in Krebs solutioncontaining 10% DMSO (Haan and Bowen, 1981). Samples werestored at -80°C and for analysis, slices were thawed rapidly andrinsed in fresh oxygenated Krebs solution before preparation oftissue for analysis.

Release of Glutamate

Release was assessed in samples of P2 prepared from untetanizedand tetanized slices that were frozen as described above or in freshlyprepared P2 according to a method described previously (McGa-hon and Lynch, 1998). In both cases, samples of synaptosomaltissue were resuspended in ice-cold Krebs solution containing 2mM CaCl2, aliquoted onto Millipore filters (0.45 �m) and rinsedunder vacuum. Tissue was incubated in 250 �l oxygenated Krebs

solution at 37°C for 3 min and filtrate was collected and stored.Release of transmitter was stimulated by the addition of 40 mMKCl to Krebs solution. In some cases, genistein (50 �M) orPD98059 (2 �M) was added to assess its effect on KCl-stimulatedrelease in vitro. In these experiments, synaptosomes were preincu-bated (15 min at 37°C) in Krebs solution containing 2 mM CaCl2with added genistein or PD98059.

Glutamate was analyzed as described previously (Ordronneau etal., 1991). Glutaraldehyde (0.5% in 100 mM NaH2PO4 buffer,pH 4.5; 320 �l) was added to 96-well plates, incubated for 60 minat 37°C, and washed with 100 mM NaH2PO4 buffer. Triplicatesamples (50 �l) or glutamate standards (50 �l; 50 nM to 10 �Mprepared in 100 mM Na2HPO4 buffer, pH 8.0) were added, in-cubated for 2 h at 37°C and washed. Ethanolamine (320 �l; 0.1 Min 100 mM Na2HPO4 buffer) was added to bind any unreactedaldehydes and donkey serum (200 �l; 3% in phosphate-bufferedsaline (PBS) containing Tween-20 (0.5%; PBS-T)) was used toblock nonspecific binding. Antiglutamate antibody (raised in rab-bit; 100 �l; 1:5,000 in PBS-T; Sigma, UK) was added, incubatedovernight at 4°C, and washed with PBS-T. Anti-rabbit horseradishperoxidase (HRP)-linked secondary antibody (95 �l; 1:10,000 inPBS-T; Amersham, UK) was added, incubated for 60 min at roomtemperature and washed. 3,3�,5,5�-Tetramethylbenzidine liquidsubstrate was added as chromogen and incubation continued forexactly 60 min at room temperature. H2SO4 (4 M; 50 �l) wasadded to stop the reaction and optical densities were determined at450 nm. Values were calculated with reference to the standardcurve, corrected for protein (Bradford, 1976) and expressed as�mol glutamate/mg protein.

Assessment of 45Ca Influx45Ca influx was assessed in samples of P2 prepared from unteta-

nized and tetanized slices of dentate gyrus obtained from saline-and genistein-treated rats by a method described previously (Kellyand Lynch, 2000). P2 was resuspended in oxygenated ice-coldincubation buffer (composition in mM: NaCl, 128; KCl 4.8;KH2PO4, 1.2; MgSO4 � 7H2O, 1.2; NaHCO3, 7.5; CaCl2, 1.3;glucose 11; ascorbic acid, 0.1; Hepes, 15; disodium EDTA, 0.3)and incubated for 5 s at 37°C in buffer containing 45Ca (finalconcentration 1 �Ci/ml; spec act, 2.1 mCi/ml; Amersham, UK)�KCl). In some experiments, genistein (50 �M) was added dur-ing incubation. Reactions were stopped by addition of 1 ml ice-cold stop buffer (composition in mM: NaCl, 118; KCl 4.8;KH2PO4, 1.2; MgSO4 � 7H2O, 1.2; NaHCO3, 26; CaCl2, 1.3;glucose, 11; ouabain, 10 mM). Samples were rinsed in a filtrationmanifold and filters added to scintillation fluid for assessment ofradioactivity. Data were expressed as nmol 45Ca2�/mg protein.

Analysis of Tyrosine Kinase Activity

Tyrosine kinase activity was assessed in P2 prepared from un-tetanized and tetanized tissue obtained from saline-and genistein-treated rats by enzyme-linked immunosorbent assay (ELISA), us-ing a nonradioactive tyrosine kinase kit (Roche MolecularBiochemicals, UK). Briefly P2 was resuspended in Krebs solutioncontaining 2 mM CaCl2 and the tyrosine phosphatase inhibitor,

378 CASEY ET AL.

sodium orthovanadate (10 mM) and aliquots were added to theassay solution to start the reaction. The assay solution consisted ofpeptide substrate (10 �l; 5 �M reconstituted in PBS–BSA (1 mg/ml), assay buffer (10 �l; Tris-HCl, 50 mM; pH 7.5; C4H6O4

Mg–4H2O, 20 mM; NaF, 5 mM; EDTA, 0.2 mM; EGTA, 0.8mM; dithiothreitol, 1 mM; sodium orthovanadate, 30 �M; ATP,1 mM), ATP-Mg2� solution (10 �l; ATP, 5 mM; MgCl2, 50 mM,pH 7) and distilled water (10 �l). Samples were incubated for 1 hat room temperature. The reaction was stopped by the addition ofthe tyrosine kinase inhibitor, piceatannol (10 �l; 3 mM in 10%dimethylsulfoxide [DMSO]). Samples were placed on ice to re-duce the temperature to 4°C and centrifuged at 10,000g for 1 min,aliquots (50 �l) were added to streptavidin-coated 96-well plates,incubated for 20 min at 37°C, and washed. Peroxidase-labeledantiphosphotyrosine antibody (75 �l; 1:100 in PBS–BSA 1 mg/ml) was added, samples were incubated for 1 h at 37°C andwashed. The manufacturer’s substrate was added as chromogenand incubation continued for 45 min at room temperature to allowmaximum color development and optical densities were deter-mined at 405 nm. Values were calculated with reference to a stan-dard curve, corrected for protein (Bradford, 1976), and expressedas picomoles tyrosine kinase/mg protein.

Analysis of Calcium Channel �-SubunitPhosphorylation

P2 was prepared from untetanized and tetanized sides of dentategyri obtained from saline-treated and genistein-treated rats. Sam-ples were equalized for protein, immune complexes were preparedby incubating tissue for immunobuffer (155 �l; Triton X-100,1.25%; NaCl, 1.9 mM; Tris-HCl, 60 mM, pH 7.4; EDTA, 6 �M,pH 8; aprotonin, 10 U/ml) for 1 h at 37°C, in the presence of anantibody raised against the �1-subunit of voltage-gated calciumchannels (anti-pan �1-subunit, Alomone Laboratories, Israel;1:25). Immune complexes were separated on 7.5% sodium dode-cyl sulfate (SDS) gels and transferred onto nitrocellulose strips(225 mA for 2 h) and reacted with antiphosphotyrosine (1:160 in2% Tris-buffered saline–Tween-20 (0.1% Tween; TBS-T) Af-finiti, UK) overnight at 4°C. Nitrocellulose strips were washed andincubated for 2 h at room temperature with secondary antibody(HRP-linked anti-rabbit antibody; 1:2,000 dilution; Amersham,UK); protein complexes were visualized by ECL detection (Amer-sham, UK). Immunoblots were exposed to film overnight andprocessed using a Fuji X-ray processor. Protein bands were quan-titated by densitometric analysis.

Analysis of ERK Phosphorylation

P2 was prepared from untetanized and tetanized sides of dentategyri obtained from saline-treated and genistein-treated rats, by amethod described previously (McGahon and Lynch, 1998). Sam-ples were analyzed for protein, diluted to equalize for protein con-centration and aliquots (10�l, 1 mg/ml) were added to 10-�lsample buffer (Tris-HCl, 0.5 mM, pH 6.8; glycerol 10%; SDS,10%; �-mercaptoethanol, 5%; bromophenol blue, 0.05% w/v),boiled for 2 min and loaded onto gels (10% SDS). Proteins wereseparated by application of 30 mA constant current for 25–30 min,

transferred onto nitrocellulose strips (225 mA for 75 min) andimmunoblotted with anti-active ERK (Promega, Madison, WI;1.5:1,000 in TBS-T containing 2% nonfat dried milk) overnightat 4°C. Nitrocellulose strips were washed and incubated for 60 minat room temperature with secondary antibody (HRP-linked anti-rabbit antibody; 1:2,000; Amersham, UK) and protein complexeswere visualized by ECL detection (Amersham, UK). Immunoblotswere exposed to film overnight and processed using a Fuji X-rayprocessor. Protein bands were quantitated by densitometric anal-ysis.

Assessment of Protein Synthesis: [35S]-Methionine Labeling

Slices of entorhinal cortex were thawed rapidly (1.5-2 min) byagitation at 37°C and rinsed four times in excess fresh Krebs solu-tion. The method used for analysis of protein synthesis has beendescribed previously (Mullany and Lynch, 1997). Briefly, sliceswere preincubated at 37°C for 10 min in 500 �l oxygenated Krebssolution containing CaCl2 (2 mM), resuspended in oxygenatedKrebs solution containing CaCl2 (2 mM), ATP (3.5 mM) and[35S]-methionine (spec act, 37TBq/mmol; 0.2 �l/ml) and incu-bated for 60 min at 37°C. At the end of the incubation period,samples were placed on ice, added to TCA (50 �l; final concentra-tion, 10%), homogenized, and added to Millipore filters (pore size0.45 �m) and filtered under vacuum. Samples were washed at least10 times by addition of ice-cold 5% TCA in H2O (200 �l). Filterpapers were added to scintillant and counted for 1.5 min. Valueswere expressed as cpm/mg protein.

Analysis of CREB Phosphorylation

Slices prepared from untetanized and tetanized sides of entorhi-nal cortex of saline-treated and genistein-treated rats were homog-enized in Krebs solution containing 2 mM CaCl2. Samples wereanalyzed for protein, diluted to equalize for protein concentrationand aliquots (10 �l, 1 mg/ml) were added to 10-�l sample buffer(Tris-HCl, 0.5 mM, pH 6.8; glycerol 10%; SDS, 10%; �-mercap-toethanol, 5%; bromophenol blue, 0.05% w/v), boiled for 10 min,and loaded onto gels (14% SDS). Proteins were separated by ap-plication of 36 mA constant current for 25–30 min, transferredonto nitrocellulose strips (225 mA for 75 min) and immunoblot-ted with anti-phospho-CREB (New England BioLabs, UK;1:1,000 in 5% BSA in Tris-buffered saline–Tween (0.1% Tween-20) containing 5% BSA) overnight at 4°C. Nitrocellulose stripswere washed and incubated for 60 min at room temperature withsecondary antibody (HRP-linked anti-rabbit antibody; 1:2,500;Amersham, UK) and protein complexes were visualized by ECLdetection (Amersham, UK). Immunoblots were exposed to filmovernight and processed using a Fuji X-ray processor. Proteinbands were quantitated by densitometric analysis.

Statistical Analysis

One-way analysis of variance (ANOVA) was performed to de-termine whether there were significant differences between condi-

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tions. When this analysis indicated significance (P � 0.05 level),the post hoc Student–Newmann–Keuls test analysis was used todetermine which conditions were significantly different from each

other. In some cases the Student’s t-test for was used to establishstatistical significance.

RESULTS

As a first step in elucidating the role of tyrosine kinase in mod-ulating transmitter release, we assessed the effect of a depolarizingpulse of KCl (40 mM) on 45Ca2� influx and glutamate release (Fig.1). KCl significantly enhanced 45Ca2� influx (Fig. 1A) and gluta-mate release (Fig. 1B; P 0.05 in each case; Student’s t-test forpaired values), but both KCl-induced responses were inhibited byincubation in the presence of genistein (50 �M).

Synaptosomes were prepared from untetanized dentate gyrusand dentate gyrus that had sustained LTP after tetanic stimulation(Fig. 2A). The data indicate that addition of 40 mM KCl to theincubation medium enhanced glutamate release (Fig. 2B) to amore marked degree in synaptosomes that had been prepared fromtetanized dentate gyrus (P 0.01; Student’s t-test for paired val-ues), compared with untetanized tissue (P 0.05 in each case;Student’s t-test for paired values). Comparison of the data indi-cated that KCl-stimulated glutamate release was increased signifi-cantly in tetanized, compared with untetanized tissue (P 0.05;Student’s t-test for unpaired means), but unstimulated glutamate

FIGURE 1. Genistein inhibits KCl-stimulated [45Ca] influx andglutamate release. A: Addition of KCl (40 mM) significantly increased45Ca influx into hippocampal synaptosomes (*P < 0.05; Student’st-test for paired samples); this effect was inhibited in tissue that wasincubated in the presence of genistein. Values are the means of 12observations and expressed as nmol/mg. B: Addition of KCl (40 mM)significantly increased glutamate release in hippocampal synapto-somes (*P < 0.05; Student’s t-test for paired samples); this effect wasinhibited by preincubation in genistein (50 �M); incubation of syn-aptosomes in the presence, compared with in the absence, of genisteinsignificantly decreased both unstimulated and KCl-stimulated gluta-mate release (�P < 0.01). Values are the means of seven observationsand expressed as �mol/mg.

FIGURE 2. LTP is associated with increased 45Ca influx andglutamate release. A: Synaptosomes were prepared from ispilateraland contralateral dentate gyrus 40 min after induction of LTP unilat-erally by high-frequency stimulation (HFS) or after low-frequencystimulation (LPF). B: Addition of KCl (40 mM) significantly in-creased glutamate release in synaptosomes prepared from untetanizeddentate gyrus (*P < 0.05; Student’s t-test for paired samples); releasewas further increased in synaptosomes prepared from tetanized den-tate gyrus (**P < 0.01). Values are the means of seven observationsand expressed as �mol/mg. The effect of KCl was similar in theunstimulated dentate gyrus and in the dentate gyrus, which received

low-frequency stimulation (E); values are means of six replicate ex-periments. C: Addition of KCl (40 mM) significantly increased [45Ca]influx into synaptosomes prepared from untetanized dentate gyrus(*P < 0.05; Student’s t-test for paired samples); influx was furtherincreased in synaptosomes prepared from tetanized dentate gyrus(**P < 0.01). These changes represent KCl-induced increases of150.28% (�11.5) and 197.04 (�26) in untetanized and tetanizedtissue, respectively (D); statistical analysis indicated that there was nosignificant difference between these values. Values are the means of 10observations and expressed as nmol/mg.

380 CASEY ET AL.

release was not altered by tetanic stimulation. The addition of 40mM KCl to the incubation medium also enhanced 45Ca2� influxin untetanized (P 0.05; Student’s t-test for unpaired means) andtetanized (P 0.01; Student’s t-test for unpaired means; Fig. 2C)tissue; KCl increased calcium influx by 150.28% (�11.5) and197.04% (�26.9) in untetanized and tetanized tissue respectively(Fig. 2D). Comparison of the percentage changes or the actualvalues for KCl-stimulated 45Ca2� influx showed no statistical sig-nificance between the values. However, an LTP-associated de-crease in unstimulated 45Ca2� influx was observed (P 0.05;Student’s t-test for unpaired means). The data indicated that ad-dition of KCl (40 mM) to the incubation medium increased glu-tamate release to a similar extent in synaptosomes prepared fromdentate gyrus of rats that received low-frequency stimulation andsynaptosomes prepared from unstimulated tissue (Fig. 2E). Inthese rats, the mean percentage change in excitatory postsynapticpotential (EPSP) slope during the last 5 min of the experiment,compared with the mean value in the time period that corre-sponded to the pre-tetanus period in the group of rats that receivedhigh-frequency stimulation, was 97.5 (�3.43, SEM; Fig. 2A).

The mean population EPSP slope in the 10 min before tetanicstimulation was 1.25 mV/ms (�0.13) in saline-treated rats; intra-cerebroventricular injection of genistein resulted in a decrease inmean EPSP slope to 0.85 mV/ms (�0.26). The data also show thatintracerebroventricular injection of genistein (250 �M) inhibitedexpression of LTP in dentate gyrus (Fig. 3A); the mean percentageincreases in population EPSP slope during the 2 min immediatelyafter tetanic stimulation, compared with the mean value in the 5min immediately before tetanic stimulation were 143.73 (�5.76,SEM) and 115.40 (�2.74) in saline-pretreated and genistein-pre-treated rats, respectively. The corresponding mean values in thelast 5 min of the experiment were 130.08 (�1.58) and 107.87(�2.11). A total of 12 saline-treated and 12 genistein-treated ratswere analyzed. At the end of the recording period, untetanized andtetanized dentate gyri were retained for subsequent preparation ofsynaptosomes for analysis; tissue from at least six rats was used ineach assay.

Figure 3B indicates that incubation of tissue in the presence ofKCl increased tyrosine kinase activity in untetanized and tetanizedtissue; this effect was statistically significant in synaptosomes pre-pared from dentate gyrus that had sustained LTP (P 0.05; Stu-dent’s t-test for paired values) but, although the increase was of asimilar order in synaptosomes prepared from untetanized dentategyrus, the increase was not statistically significant due to the verylarge intersample variability. Pretreatment with genistein pre-vented the KCl-stimulated release in tetanized samples and Figure3C demonstrates that while KCl significantly increased glutamaterelease in synaptosomes prepared from untetanized dentate gyrusof saline-pretreated rats (P 0.05; ANOVA), release was en-hanced to a greater extent in synaptosomes prepared from teta-nized dentate gyrus (P 0.01; ANOVA). Both unstimulated andKCl-stimulated glutamate release were decreased in synaptosomesprepared from the dentate gyrus of genistein-pretreated rats;genistein pretreatment also inhibited the stimulatory effect of KClon glutamate release. Similar data were obtained in both unteta-nized and tetanized tissue.

Figure 4A shows one sample immunoblot that indicates thattyrosine phosphorylation of the calcium channel �-subunit wasincreased in tetanized tissue prepared from saline-treated, but not

FIGURE 3. Genistein pretreatment inhibits LTP and the LTP-associated increases in tyrosine kinase and glutamate release. A: In-tracerebroventricular injection of genistein (250 �M) 40 min beforetetanic stimulation markedly attenuated LTP in dentate gyrus. B:Addition of KCl (40 mM) increased tyrosine kinase activity in synap-tosomes prepared from untetanized dentate gyrus of saline-treatedrats, but this did not reach statistical significance due to the largevariability; a significant increase was observed in tetanized tissue(*P < 0.05; Student’s t-test for paired samples). Unstimulated ty-rosine kinase activity in synaptosomes prepared from tetanized tissuewas significantly increased compared with untetanized tissue (�P <0.05; Student’s t-test for paired samples). KCl failed to increase kinaseactivity in untetanized or tetanized tissue prepared from genistein-treated rats. Values are the means of 6 observations and expressed asnmol/mg. C: Addition of KCl (40 mM) increased glutamate release insynaptosomes prepared from untetanized dentate gyrus of saline-treated rats (*P < 0.05; Student’s t-test for paired samples); KCl-stimulated release was more marked in synaptosomes prepared fromtetanized dentate gyrus (**P < 0.01). Unstimulated release was sig-nificantly reduced in synaptosomes prepared from both tetanized anduntetanized tissue of genistein-treated rats (�P < 0.01; Student’st-test for unpaired samples) and KCl failed to increase release in thesepreparations. Values are the means of 6 observations and expressed as�mol/mg.

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genistein-treated, rats. Densitometric analysis of the individual ex-periments allowed assessment of the mean changes and showedthat tyrosine phosphorylation of the calcium channel �-subunitwas significantly enhanced in synaptosomes prepared from teta-nized, compared with untetanized, dentate gyrus of saline-treated

rats (P 0.05; ANOVA). This LTP-associated increase in phos-phorylation was inhibited by genistein pretreatment (Fig. 4A).

While ERK phosphorylation was not affected by low-frequencystimulation as shown by the mean data obtained after densitomet-ric analysis (Fig. 4C), Figure 4B shows one sample immunoblotthat demonstrates that ERK phosphorylation was increased in tet-anized tissue prepared from saline-treated, but not genistein-treated rats. Densitometric analysis revealed that mean ERK phos-phorylation was increased significantly in synaptosomes preparedfrom tetanized, compared with untetanized, dentate gyrus of sa-line-pretreated rats (P 0.05; ANOVA), but that this effect wasinhibited in tissue prepared from genistein-treated rats. The dataalso showed that ERK phosphorylation was significantly decreasedin synaptosomes prepared from untetanized tissue of genistein-treated, compared with saline-treated rats (P 0.05; ANOVA;Fig. 4B). The finding that PD98059, which inhibits ERK phos-phorylation, blocked KCl-stimulated glutamate release in vitro(Fig. 4D) suggests that depolarization-induced release is modu-lated by ERK activity.

We analyzed protein synthesis in the cell bodies of the perforantpath synapses, i.e. in the entorhinal cortex. Figure 5B indicates that35S-methionine incorporation into proteins was significantly en-hanced in slices prepared from entorhinal cortex obtained from thetetanized, compared with the untetanized, side of the brain ofsaline-treated rats (P 0.05; ANOVA). Protein synthesis wasdecreased with genistein treatment (P 0.05; ANOVA) and, inthese samples, there was no evidence of the enhanced protein syn-thesis associated with tetanization. In parallel with the LTP-asso-ciated increase in protein synthesis, we observed an LTP-associatedincrease in phosphorylation of CREB. Figure 5A shows one sampleimmunoblot; the mean data were derived from densitometric anal-ysis showing that CREB phosphorylation was significantly en-hanced in cells prepared from entorhinal cortex obtained from thetetanized (compared with untetanized) side of the brain of saline-treated rats (P 0.05; ANOVA). Figure 5A also shows thatgenistein inhibited the LTP-associated increase in CREB phos-phorylation. Neither protein synthesis nor CREB phosphorylationin entorhinal cortex were affected by low-frequency stimulation tothe perforant path (Fig. 5A,B).

DISCUSSION

We set out to investigate the possibility that tyrosine kinaseactivity exerted an impact on LTP in perforant path–granule cellsynapses by modulating glutamate release. We report that inhibi-tion of tyrosine kinase by intracerebroventricular injection ofgenistein, blocked expression of LTP and the LTP-associated in-creases in glutamate release and calcium influx. The data indicatethat LTP was accompanied by increases in phosphorylation of the�1-subunit of voltage-sensitive calcium channels and ERK in pre-synaptic terminals and increased phosphorylation of CREB in theentorhinal cortex.

FIGURE 4. Genistein inhibits the LTP-associated increases inphosphorylation of the �1-subunit of voltage sensitive calcium chan-nels and ERK. A: The sample immunoblot indicates that phosphory-lation of the �1-subunit of voltage sensitive calcium channels wasincreased in synaptosomes prepared from tetanized (lane 2), com-pared with untetanized (lane 1) dentate gyrus of saline-treated rats.Densitometric analysis of data from six separate experiments, given inarbitrary units, indicate that the difference was statistically significant(*P < 0.05; Student’s t-test for paired samples). This effect was inhib-ited in tissue prepared from genistein-pretreated rats as shown on thesample immunoblot (cf. lanes 3, 4) and by analysis of the mean valuesobtained by densitometric analysis. B: The sample immunoblot indi-cates that phosphorylation of ERK was increased in synaptosomesprepared from tetanized (lane 2), compared with untetanized (lane 1),dentate gyrus of saline-treated rats. Densitometric analysis of datafrom six separate experiments, given in arbitrary units, indicate thatthe difference was statistically significant (*P < 0.05; Student’s t-testfor paired samples). This effect was inhibited in tissue prepared fromgenistein-pretreated rats as shown on the sample immunoblot (cf.lanes 3, 4) and by analysis of the mean values obtained by densito-metric analysis. C: ERK phosphorylation was similar in unstimulatedtissue and tissue prepared from dentate gyrus that received low-fre-quency stimulation. D: Addition of KCl (40 mM) to the incubationmedium significantly increased glutamate release in synaptosomesprepared from dentate gyrus (**P < 0.01; Student’s t-test for pairedsamples); this effect was attenuated when PD98059 (2 �M) was in-cluded in the incubation. Values are means of 6 experiments andexpressed as �mol glutamate/mg protein.

382 CASEY ET AL.

As a first step in analyzing the effect of tyrosine phosphorylationon synaptic function in hippocampus, we assessed the effect ofgenistein on endogenous glutamate release and calcium influx insynaptosomes prepared from hippocampal synaptosomes and ob-served that depolarization-induced increases in both measures wereinhibited by genistein. This suggests that tyrosine phosphorylationof one or more proteins is required to permit a depolarizing pulseof KCl to stimulate calcium entry and to release glutamate. Theinhibitory effect of genistein on endogenous glutamate release isconsistent with earlier findings (Mullany et al., 1996; Phillis et al.,1996).

The present data also argue for a role for tyrosine kinase in LTPin dentate gyrus (McGahon and Lynch, 1998) and CA1 in whichactivation of fyn (Grant et al., 1992; Kojima et al., 1997; Lu et al.,1999) and src (Lu et al., 1998; Huang and Hsu, 1999) were cou-pled with LTP. The present data do not assist in identifying thefamily of tyrosine kinases that are activated after tetanic stimula-tion, but we have previously shown that LTP in dentate gyrus isaccompanied by increased tyrosine phosphorylation of the NGFreceptor, TrkA, suggesting a role for activation of cascades trig-gered by stimulation of receptor tyrosine kinases (Maguire et al.,1999).

The question of identification of the protein substrates targetedby the LTP-associated increase in tyrosine kinases arises. SinceLTP in dentate gyrus is accompanied by increased glutamate re-lease as described in the present study, and in several previousreports (Errington et al., 1987; Canevari et al., 1994; McGahon etal., 1999; Maguire et al., 1999; Kelly and Lynch, 2000), it isappropriate to consider proteins that might modulate transmitterrelease. We investigated the possibility that the �1-subunit of cal-cium channels might be a substrate for tyrosine kinase because (1)the in vitro data presented suggested that calcium influx was sen-sitive to tyrosine kinase activity; (2) calcium channel activity mod-ulates calcium influx, which in turn modulates transmitter release;and (3) several reports have indicated that calcium channel activityis altered by tyrosine phosphorylation (Arnoult et al., 1997; Potierand Rovira, 1999; Wijetunge et al., 2000; Strauss et al., 2000).Our findings show that tyrosine phosphorylation of the �1-sub-unit of calcium channels was markedly increased in synaptosomesprepared from tetanized dentate gyrus and this effect was inhibitedby genistein. One consequence of a phosphorylation-induced in-crease in calcium influx is likely to be an increase in glutamaterelease; the present data that show parallel changes in tyrosinephosphorylation of �1-subunit of calcium channels, calcium in-flux and release after induction of LTP, and the parallel inhibitoryeffects of genistein suggest a possible causal relationship betweenthese synaptic events. In the context of these findings, it is signifi-cant that an NMDA-independent form of LTP, described in CA1both in vitro and in vivo, is dependent on activation of voltage-sensitive calcium channels (Grover and Teyler, 1994; Morgan andTeyler, 1999). Interestingly, the tyrosine kinase inhibitors,genistein and lavendustin A blocked this form of LTP, but not theNMDA-dependent form (Cavus and Teyler, 1996).

The mitogen-activated protein kinases, including ERK, are ac-tivated by dual phosphorylation on threonine and tyrosine resi-dues. Several regulators of ERK have been identified, includinggrowth factors and neurotransmitters, but activation by calciumand tyrosine kinase have also been reported (see Derkinderen et al.,1999). In this study, we observed an LTP-coupled increase in ERKactivation, coincident with increased tyrosine kinase activity andincreased calcium influx and parallel inhibition of these functionsin tissue prepared from genistein-treated rats. The coupling ofthese events permits us to suggest that a causal relationship existsbetween them, and that the activation of ERK described in thepresent work might be stimulated by tyrosine kinase acting di-rectly, or indirectly as a consequence of increased calcium influx.However we have previously found that LTP is associated with

FIGURE 5. Genistein inhibited the LTP-associated increases inCREB phosphorylation and protein synthesis in entorhinal cortex. A:The sample immunoblot indicates that phosphorylation of CREB wasincreased in homogenate prepared from entorhinal cortex obtainedfrom the tetanized side of the brain of saline-treated rats (lane 2),compared with the untetanized side (lane 1). Densitometric analysisof data from six separate experiments, given in arbitrary units, indi-cate that the difference was statistically significant (*P < 0.05; Stu-dent’s t-test for paired samples). This effect was inhibited in tissueprepared from genistein-pretreated rats as shown on the sample im-munoblot (cf. lanes 3, 4) and by analysis of the mean values obtainedby densitometric analysis. B: 35S-Methionine labeling of proteins wassignificantly increased in slices of entorhinal cortex prepared from thetetanized side of the brain of saline-treated rats, compared with theuntetanized side (*P < 0.05; Student’s t-test for paired samples). Thiseffect was inhibited in tissue prepared from genistein-pretreated rats.Low-frequency stimulation did not significantly alter either CREBphosphorylation or protein synthesis.

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increased release of both NGF (Kelly et al., 2000) and BDNF(Gooney et al., 2000) from slices of dentate gyrus, and that they acton presynaptic TrkA and TrkB, respectively; it is possible that theseneurotrophins contribute to activation of ERK observed in thepresent study. Other modulators of ERK include PI3-kinase (e.g.,Lopez-Ilasaca et al., 1997), phosphorylation of which is increasedwith LTP (Kelly and Lynch, 2000), and the small GTP-binding pro-tein, ras (acting through the ras/raf/MEK cascade), which may beactivated by tyrosine kinase-induced stimulation of the adaptor pro-tein, SHC (see Graves and Krebs, 1999). One consequence of in-creased ERK phosphorylation may be upregulation of glutamate re-lease since the data presented provide evidence that inhibition of ERKexerted an inhibitory effect on stimulus-dependent glutamate release.

One downstream consequence of an increase in ERK activity isan increase in activation of the transcription factor, CREB (Fink-beiner, 2000), which is also activated by protein kinase A (Nguyenand Kandel, 1996) and Ca2�/calmodulin-dependent kinase(Finkbeiner et al., 1997). Recent data have indicated that phos-phorylation of CREB may be a necessary step in inducing mor-phological changes stimulated by estradiol in hippocampal neu-rons (Murphy and Segal, 1997). In support of such a role forCREB, a coupling between increased CREB phosphorylation andprotein synthesis are considered to be vital cell responses in thestabilization of long-term facilitation in Aplysia (Casadio et al.,1999), while CREB phosphorylation also seems to mediateBDNF-induced transcription (Finkbeiner et al., 1997). The datapresented provide direct evidence that tetanically induced LTP inperforant path–granule cell synapses is coupled with increasedCREB phosphorylation in entorhinal cortex and that this change isaccompanied by increased protein synthesis. While these observa-tions reflect presynaptic changes, increased CREB phosphoryla-tion has been reported postsynaptically after induction of LTP indentate gyrus (Schultz et al., 1999; Davis et al., 2000) and CA1(Matthies et al., 1997), in which an increase in phospho-CREBimmunofluorescence was also observed after stimulation of theSchaffer collaterals (Lu et al., 1999). Despite some experimental dif-ferences, it is significant that the timing of the response in the presentstudy was similar to that observed elsewhere (Schultz et al., 1999).

The fact that increased protein synthesis accompanied increasedCREB phosphorylation after induction of LTP, and that botheffects were inhibited when LTP was blocked, is consistent withthe view that they are causally related. It is tempting to propose thatphosphorylation of CREB is the first of a number of changes thatstimulate synthesis of specific proteins, which in turn might or-chestrate the morphological changes required for the more persis-tent aspects of LTP (Lisman and Harris, 1993; Edwards, 1995). Inthe context of the present study it is significant that the cAMP-induced synaptic plasticity in Drosophila neuromuscular junctionis a consequence of a CREB-mediated increase in transmitter re-lease (Davis et al., 1996). On the basis of this latter observation, wemight speculate that CREB phosphorylation might contribute tothe LTP-associated increase in glutamate release.

The focus of the present study was to assess changes that occurpresynaptically after induction of LTP in dentate gyrus that mightplay a role in modulating glutamate release. We did not addresschanges in the postsynaptic cell. However it must be acknowledged

that the data correlate LTP with biochemical changes, for example,increases in release and protein synthesis; the fact that thesechanges are inhibited when LTP is blocked as shown in the presentwork, or by the NMDA receptor antagonist, AP5 (Mullany andLynch, 1997, 1998), provides strong evidence of a causal relation-ship between the measures. The data presented support the viewthat activation of tyrosine kinase, one consequence of tetanic stim-ulation, stimulates the increase in glutamate release which accom-panies LTP. It is likely that this is achieved by phosphorylating anumber of proteins that include the �1-subunit of voltage-sensi-tive calcium channels, phosphorylation of which leads to enhancedcalcium influx. It appears reasonable to suggest that activation ofERK, which is inhibited when tyrosine kinase activity is inhibited,contributes to the enhancement of release and to phosphorylationof CREB, which in turn may trigger synthesis of specific proteinsthat are likely to underpin the more persistent components of LTP.

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