Signaling mechanisms mediating BDNF modulation of memory formation in vivo in the hippocampus

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Cellular and Molecular Neurobiology [cemn] pp690-cemn-456076 December 14, 2002 19:31 Style file version Oct 23, 2000

Cellular and Molecular Neurobiology, Vol. 22, Nos. 5/6, December 2002 ( C© 2002)

Signaling Mechanisms Mediating BDNF Modulationof Memory Formation In Vivo in the Hippocampus

Mariana Alonso,1 Monica R. M. Vianna,2 Ivan Izquierdo,2 and Jorge H. Medina1,3

Received June 14, 2002; accepted September 20, 2002

SUMMARY

Given that brain-derived neutrophic factor (BDNF) modulates both short-term synap-tic function and activity-dependent synaptic plasticity in the adult hippocampus, here weexamined signaling mechanisms in vivo in the hippocampus mediating BDNF modulation oflong-term memory (LTM) formation of a one-trial fear-motivated learning task in rats. Bi-lateral infusions of function-blocking anti-BDNF antibody into the CA1 region of the dorsalhippocampus decreased extracellular-signal regulated kinase 2 (ERK2) and CREB activa-tion and impaired LTM retention scores. Inhibition of ERK1/2 activation by PD098059produced similar effects and also reduced CREB phosphorylation. In contrast, intrahip-pocampal administration of recombinant human BDNF increased ERK1/2 and CREB ac-tivation and facilitated LTM. Activated-p38, activated-PKC isoforms, and activated-AKTwere unaltered after BDNF or anti-BDNF infusion. In addition, no changes were foundon αPKA and βPKA catalytic subunits in nuclear samples. Thus, our results suggest thatBDNF exerts its role in LTM formation in vivo in CA1 region of the hippocampus, at leastin part, via CREB activation. Moreover, BDNF-induced CREB activation appears to bemediated mainly through the activation of ERK1/2 signaling pathway.

KEY WORDS: endogenous BDNF; anti-BDNF antibody; memory consolidation;inhibitory avoidance learning; signaling pathways; ERK1/2; CREB.

INTRODUCTION

Brain-derived neutrophic factor (BDNF) plays diverse roles in regulating neuronalstructure, function, and long-term survival of specific populations of neurons in thedeveloping and adult brain, and it is involved in activity-dependent modulation ofdendritic and axonal growth (Lu and Chow, 1999; McAllister et al., 1999; Poo, 2001;Schinder and Poo, 2000).

A growing body of evidence indicates that BDNF regulates both short-termsynaptic function and long-term activity-dependent synaptic plasticity (Kafitz et al.,1999; Korte et al., 1998; Li et al., 1998; Rutherford et al., 1998; Tyler and Pozzo-Miller,

1 Instituto de Biologia Celular y Neurociencias, Facultad de Medicina, UBA, Buenos Aires, Argentina.2 Centro de Memoria, Departamento de Bioquimica, Instituto de Biociencias, UFRGS, Porto Alegre,

Brazil.3 To whom correspondence should be addressed at Instituto de Biologia Celular y Neurociencias, Facultad

de Medicina, UBA, Paraguay 2155, piso 3, 1121 Buenos Aires, Argentina; e-mail: [email protected].

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0272-4340/02/1200-0663/0 C© 2002 Plenum Publishing Corporation

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664 Alonso, Vianna, Izquierdo, and Medina

2001; see for references McAllister et al., 1999; Poo, 2001). It has been shown thatBDNF acutely modulates both pre- and postsynaptic aspects of synaptic transmission(see Poo, 2001) and it is crucially involved in long-term potentiation (LTP) (Chenet al., 1999; Figurov et al., 1996; Kang et al., 1997; Korte et al., 1998; Kossel et al., 2001;Patterson et al., 2001; Poo, 2001).

The finding that BDNF plays an important role in the induction and/or mainte-nance of hippocampal LTP and that CREB (cyclic AMP response element-bindingprotein), a transcription factor involved in long-term memory (LTM) formation andsynaptic plasticity, is a major mediator of neuronal BDNF responses (Finkbeineret al., 1997) fueled the study of the role of this neurotrophin in learning and memory(for review see Yamada et al., 2002). In this regard, intracerebral pretraining admin-istration of antisense BDNF oligonucleotides impaired LTM formation in spatial(Mizuno et al., 2000) and avoidance learning tasks (Johnston and Rose, 2001; Maet al., 1998). Pretraining infusion of BDNF antibodies caused amnesia for a spa-tial task (Mu et al., 1999) in rats and for an inhibitory avoidance in day-old chicks(Johnston et al., 1999). Transgenic mice overexpressing BDNF showed deficits inLTM (Croll et al., 1999). In contrast, BDNF mutant mice exhibited learning, but notmemory retention, deficits of a multitrial spatial task (Linnarsson et al., 1997). In ad-dition, acute or chronic intracerebral administration of BDNF produced no effectson memory retention (Cirulli et al., 2000; Fischer et al., 1994; Pelleymounter et al.,1996; but see also Johnston et al., 1999).

Recently, we demonstrated that hippocampal BDNF is required for both short-term memory (STM) and LTM formation of an inhibitory avoidance training (Alonsoet al., 2002). Additionally, our results suggest that this requirement involves ERK1/2-dependent and -independent mechanisms.

Studies using cell cultures and/or acute or cultured microslices (Blanquet, 2000;Finkbeiner et al., 1997; Gottschalk et al., 1999; Pizzorusso et al., 2000) have iden-tified a number of important signaling pathways that are activated by BDNF andother members of the neurotrophic factor family. These pathways are initiated bythe binding of neurotrophins to tyrosine kinase receptor (TRK) leading to receptorautophosphorylation at specific tyrosine residues within the intracellular domains(Soppet et al., 1991; for review see Kaplan and Stephens, 1994). The phosphory-lated tyrosines serve as protein interaction sites for SHC, phospholipase C-γ (PLC-γ ), and phosphotidylinositol-3 kinase (PI3K), the intracellular molecules that leadto the activation of three major signaling pathways (Blanquet, 2000; Gottschalket al., 1999; Stephens et al., 1994). Tyrosine phosphorylation of SHC then triggersSHC/Grb2/Sos interaction, Ras activation, and a series of phosphorylation reac-tions that include Raf, mitogen-activated protein kinase (MEK), and extracellularregulated protein kinases (ERK1/2) (for review see Segal and Greenberg, 1996).The active PLC-γ cleaves phosphotidylinositol-4,5-biphosphate to generate inosi-toltrisphosphate (IP3) and diaylglycerol (DAG), which in turn induces the releaseof Ca2+ from internal stores and activates protein kinase C (PKC), respectively (forreview see Kaplan and Stephens, 1994). Release of Ca2+ from internal stores alsoactivates calcium/calmodulin-dependent protein kinase-2 (CAMKII) and p38 sub-family of mitogen-activated protein kinases in hippocampal neurons (Blanquet, 2000;

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Endogenous BDNF, Signaling Mechanisms and Memory 665

Blanquet and Lamour, 1997). Finally, the putative effector of PI3K is the serine andthreonine kinase Akt (Burgering and Coffer, 1995; Franke et al., 1995).

Given that signaling mechanisms mediating the in vivo actions of BDNF onmemory formation remain largely unknown, in the present study we examined theBDNF-triggered events involved in LTM modulation by intrahippocampal microin-fusion of function-blocking anti-BDNF antibody or recombinant BDNF in the CA1region.

Our results demonstrated that hippocampal BDNF activates in vivo ERK1/2/CREB in the hippocampal CA1 region. The regulation of this signaling pathwayappears to be associated with some of the modulatory effects of BDNF on LTMformation.

MATERIALS AND METHODS

Subjects

Male Wistar rats (2–3 months; weight 160–250 g) from our own breeding stockwere used. The animals were housed in plastic cages with water and food available adlibitum, under a 12 h light/dark cycle (light on at 7:00 A.M.) at a constant temperatureof 23◦C.

Behavioral Procedure

Inhibitory avoidance was as follows (Bernabeu et al., 1997; Izquierdo et al., 1998).Rats were placed on a 2.5 cm high, 8.0 cm wide platform at left of a 50.0× 25.0×25.0 cm yellow acrylic training apparatus, whose floor was a series of parallel 0.2 cmcaliber bronze bars spaced 1.0 cm apart. Latency to step down onto the grid withall four paws was measured. In the training trial, immediately after this, the animalsreceived a 0.4 mA, 4.0 s scrambled foot shock. In the test session performed 24 hafter training, the procedure was similar except that foot shock was omitted.

Surgery and Infusion Procedures

Male Wistar rats were implanted under deep thionembutal anesthesia with 30ga guide cannulae in the dorsal CA1 region of the hippocampus at the coordinatesof the atlas of Paxinos and Watson (1986): anterior, −4.3; lateral, ±2.8; ventral,2.6. The cannulae were fixed to the skull with dental acrylic (Bernabeu et al., 1997;Izquierdo et al., 1998). After recovery from the surgery, these animals were trained ininhibitory avoidance and tested 24 h later. Cannulated rats received 3 h after training,a bilateral infusion of saline or recombinant human BDNF (0.25 µg/side, Promega,Madison, WI) or function-blocking anti-BDNF antibody (0.5 µg/side, Chemicon,Temecula, CA, AB1513P). Infusions were in all cases bilateral and had a volume of0.8 µL (except for the experiments with PD 098059 where it had 0.5 µL). The entireinfusion procedure took ≈2 min, including 45 s for the infusions themselves, firston one side and then on the other, and the handling. Histological examination of

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Fig. 1. Schematic drawing of plane AP: −4.3 modified from the atlas ofPaxinos and Watson (1986), showing (strippled) the extend of the areareached by the infusion in the dorsal hippocampus.

cannulae placements was performed as described previously (Bernabeu et al., 1997;Izquierdo et al., 1998) (see Fig. 1). The behavioral data only from animals with bothcannulae located in the intended site were used.

Immunoblot Assays

To investigate whether intrahippocampal infusion of recombinant BDNF oranti-BDNF antibody or PD098059 (50 µM, RBI, Natick, MA), a specific MEK in-hibitor, affects different signaling pathways, 3-mm-thick slices were taken 5–7 minafter the infusions from the area in which the infusion cannulae were placed. Tis-sue was homogenized in ice-chilled buffer (20 mM Tris-HCL, pH 7.4, 0.32 M su-crose, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 10 µg/mL aprotinin, 15 µg/mLleupeptin, 50 mM NaF, and 1 mM sodium orthovanadate). To evaluate levels ofpCREB and nuclear translocation of PKA catalytic subunits, nuclear pellets wereprepared as described previously (Viola et al., 2000). Samples of homogenates (16–30 µg of protein) or nuclear fraction (25 µg) were subjected to SDS-PAGE (10%gels), and immunoblots were performed as described previously (Cammarota et al.,2000; Viola et al., 2000). Membranes were incubated with the following antibodies:anti-ERK1 and ERK2 (1:3000; New England Biolabs, Beverly, MA), anti-activatedERK1 and ERK2 (1:4000; New England Biolabs), anti-activated p38 (1:1000, NewEngland Biolabs), anti-activated AKT (1:2000; New England Biolabs), anti-activatedCAMKII (1:5000; Promega), anti-activated pan-PKC (1:5000, New England Biolabs),anti-CREB (1:1000, New England Biolabs), anti-activated CREB (1:1500, New Eng-land Biolabs), and anti-α PKA catalytic subunit (1:5000, Santa Cruz). Densitometricanalysis of the films was performed by using an MCID Image Analysis System (ver-sion 5.02, Imaging Research, Inc., St. Catharines, Ontario, Canada). Western blotswere developed to be linear in the range used for densitometry.

Data Analysis

Statistical analysis was performed by one-way ANOVA using Newman Keulstests. Mann–WhitneyU test was used for nonparametric analysis after Kruskal–Wallistest.

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RESULTS

We have recently demonstrated that hippocampal BDNF is required for LTMformation (Alonso et al., 2002). LTM was completely disrupted when function-blocking anti-BDNF antibody was infused into CA1 region of the dorsal hippocam-pus 15 min prior, or 1 and 4 h after training rats in a one-trial inhibitory avoidancetask. However, when administered immediately or 6 h after training, anti-BDNFantibody had no effect on retention test performance. In contrast, infusion ofrecombinant BDNF into CA1 region 1 and 4 h posttraining facilitated LTMretention.

Extending these findings we found here that the infusion of function-blockinganti-BDNF 3 h after training caused a full amnesia as tested 24 h after training(Fig. 2). Also, infusion of recombinant BDNF at the same time point facilitated LTMretention. These results indicate that endogenous BDNF was also required for LTMformation 3 h after training.

Given that all the studies on BDNF modulation of CA1 hippocampal synapseshave been performed on cell cultures and/or acute or cultured microslices (Blanquet,2000; Finkbeiner et al., 1997; Gottschalk et al., 1999; Pizzorusso et al., 2000), it isimportant to examine the BDNF-triggered events in vivo that could underlie thememory modulation by BDNF. Therefore, we next examined whether the infusionin vivo of recombinant BDNF or anti-BDNF antibody into the CA1 region of thedorsal hippocampus alters different signaling pathways in microdissected samples ofthis region.

Given that BDNF stimulates ERK1/2 in hippocampal and cortical neurons(Blanquet, 2000; Cavanaugh, 2001; Han and Holtzman, 2000), in CA1 microslices

Fig. 2. Hippocampal BDNF is required for long-term memory formation.Medians (interquartile range) of latencies to step down from the platformof the inhibitory avoidance box in the test session 24 h after training. An-imals were infused with saline, or BDNF (0.25 µg/side) or anti-BDNF(0.5 µg/side), 3 h after avoidance training. ∗∗∗ p < 0.001, ∗∗ p < 0.01 versussaline group; Mann–Whitney U test, n = 9–10 per group.

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(Gottschalk et al., 1999), and in dentate gyrus in vivo (Ying et al., 2002) we firstdetermined whether the infusion of recombinant BDNF or anti-BDNF antibodymodulates the levels of activated ERK1/2 in the CA1 region.

Using immunoblot techniques to detect dually phosphorylated, activatedERK1/2 we found that the infusion of recombinant BDNF (0.25µg/side) produced anincrease in the phosphorylation state of ERK1/2 (Fig. 3(A)) and that the administra-tion of anti-BDNF antibody (0.5µg/side) decreased ERK2, but not ERK1, activation

Fig. 3. In vivo memory modulation by BDNF is associated with ERK1/2 activation in the CA1 region.(A). Representive Western Blots and densitometric analysis of the data using anti-activated ERK1/2(P-ERK1/2) in the area of the dorsal hippocampus in which the infusion cannulae were placed, 5–7 minafter animals were injected with saline or BDNF (0.25 µg/side) or anti-BDNF (0.5 µg/side) or PD 098059(50µM). Data are expressed as mean± SEM of O.D. in arbitrary unit, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05with respect to saline values, Newman–Keuls test, n = 4–6. (B–E). Same as in (A) but using antiactivatedp38 (P-p38, B), antiactivated AKT (P-AKT, C), antiactivated CAMKII (P-CAMKII, D), and antiactivatedpan-PKC (P-α/βI/IIPKC and P-δ PKC, E). Data are expressed as mean± SEM of O.D. in arbitrary unit.In (D), ∗ p < 0.05 with respect to saline values, Newman–Keuls test, n = 5–6.

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(Fig. 3(A)). When blots were stripped and reprobed using an antibody that recognizesERK1/2 independently of their phosphorylation states (total ERK1/2), no changeswere observed (ERK1: saline: 73.1± 26.8, BDNF: 51.8± 22, anti-BDNF: 70.3± 12;ERK2: saline: 811.09± 151.3, BDNF: 810.5± 299, anti-BDNF: 1313.6± 296, n = 5–6, p > 0.05). Given that we previously demonstrated that the infusion of PD098059,a ERK1/2 kinases inhibitor, administered 3 h but not 0, 1, 4 h or 6 h after train-ing impairs LTM formation (Alonso et al., 2002; Walz et al., 1999), we next deter-mined whether PD098059 effectively inhibits ERK1/2 activation in vivo. As shownin Fig. 3(A), PD098059 (50 µM/side) caused a significant reduction in ERK2 activa-tion (−62%, p < 0.01, n = 5). No changes were observed in total ERK1/2 (ERK1:(102.7± 47.9)% with respect to mean saline values; ERK2: (114.1± 25.5)%, n = 5–6, p > 0.05). It is important to note here that PD 098059 had no effect on CAMKIIor AKT activation (n = 4, data not shown).

These findings suggest that ERK1/2 is a downstream component of the signalingpathway mediating the effect of endogenous BDNF on LTM formation at a precisetime window (3 h after training).

We have demostrated that ERK1/2-independent signaling pathways are alsoinvolved in BDNF modulation of LTM formation (Alonso et al., 2002). In this con-text, we next determined whether the infusion of recombinant BDNF or anti-BDNFantibody alters the activation levels of several major BDNF-triggered signalingpathways.

In contrast with previous results obtained in culture neurons and/or slices(Blanquet, 2000; Gottschalk et al., 1999), no changes were found on p38, AKT,α/βI/IIPKC, or δPKC activation after BDNF or anti-BDNF infusions with respect to saline-infused controls (Fig. 3 (B), (C), and (E)), suggesting that these cascades are notinvolved in rapid BDNF-triggered events in vivo in the CA1 region.

Interestingly, in the same conditions, the in vivo infusion of BDNF caused asignificant reduction on CAMKII activation in the CA1 region (Fig. 3(D)) but nochanges were observed after anti-BDNF antibody infusion. These results suggesta rapid pharmacological action of exogenous recombinant BDNF on the levels ofactivated CAMKII.

Based on our findings demonstrating that hippocampal cAMP/PKA cascadeactivation is required for LTM formation (Bernabeu et al., 1997; Bevilaqua et al., 1997;Vianna et al., 1999), we decided to assess whether the infusion of recombinant BDNFor anti-BDNF antibody modifies the nuclear translocation of catalytic subunits ofPKA (PKAc). Using an antibody that recognizes theαPKAc andβPKAc subunits, nochanges were found in the amount of these two PKAc subunits in nuclear fractionsafter BDNF or anti-BDNF infusions (fig. 4(A)). This finding suggests that PKAactivation and subsequent nuclear translocation is not involved in rapid signalingBDNF-triggered events in vivo in the CA1 region.

Evidence suggest that activation of CREB is a crucial step for memory pro-cessing of a one-trial inhibitory avoidance training in rats (Bernabeu et al., 1997;Bourtchuladze el al., 1994; Taubenfeld et al., 1999). In addition, Finkbeiner et al.(1997) have shown that BDNF in cultures, neurons, and hippocampal slices stim-ulates CREB phosphorylation in CA1 region acting as an important regulator ofBDNF-induced gene expression.

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Fig. 4. In vivo memory modulation by BDNF is associated with CREB activation in theCA1 region. (A). Representive Western Blots and densitometric analysis of the data usinganti-PKAc (αPKAc and βPKAc) in nuclear samples from area of the dorsal hippocampusin which the infusion cannulae were placed, 5–7 min after animals were injected with salineor BDNF (0.25 µg/side) or anti-BDNF (0.5 µg/side). Data are expressed as mean± SEMof O.D. in arbitrary unit. (B). Representive Western Blots and densitometric analysis of thedata using anti-pCREB (p-CREB) in nuclear samples from area of the dorsal hippocampusin which the infusion cannulae were placed, 5–7 min after animals were injected withsaline or BDNF (0.25 µg/side) or anti-BDNF (0.5 µg/side) or PD 098059 (50 µM). Dataare expressed as mean± SEM of O.D. in arbitrary unit, ∗ p < 0.05 with respect to salinevalues, Newman Keuls test, n = 5–6.

We next determined whether the infusion of recombinant BDNF or anti-BDNFantibody into the CA1 region modulates CREB phosphorylation levels. Using anantibody that specifically detects the levels of Ser 133 pCREB, we found that theinfusion of recombinant BDNF (0.25 µg/side) produced an increase in the levels ofpCREB (Fig. 4(B)). On the other hand, the administration of anti-BDNF antibody(0.5 µg/side) decreased the levels of pCREB (Fig. 4(B)). When blots were stripped

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and reprobed using an antibody that recognizes CREB independently of its phos-phorylation state (total CREB), no changes were observed (saline: 882.8± 163.8,BDNF: 484.1± 160, anti-BDNF: 642.5± 63.8, n = 4–5, p > 0.05).

Given that ERK activation leads to phosphorilation of CREB at Ser-133 in thehippocampus (Davis and Squire, 2000; Roberson et al., 1999; Xing et al., 1996; Yinget al., 2002) we next determine whether the inhibition of ERK1/2 by PD098059 altersthe levels of pCREB in vivo. As shown in Fig. 4(B), PD098059 (50 µM/side) causeda significant reduction in pCREB levels. No changes were observed in total CREB(113.02± 44.8)% with respect to mean saline values, n = 5, p > 0.05). Accordingto previous works with hippocampal slices (Finkbeiner et al., 1997; Roberson et al.,1999), our results suggest that BDNF/ERK1/2 signaling pathway plays a critical roleon CREB phosphorylation CA1 region of the hippocampus.

DISCUSSION

Current evidence suggest that BDNF released during memory acquisition andconsolidation periods plays an obligatory role in long-lasting memory formation ofdiverse learning tasks in the CA1 region of the dorsal hippocampus (Alonso et al.,2002; for review see Yamada et al., 2002).

The present study is, to the best of our knowledge, the first to explore BDNFmechanisms involved in LTM modulation in the intact brain. We show that locallyapplied anti-BDNF antibody 3 h after training, completely blocked LTM formationin a passive avoidance learning task. We also found that recombinant BDNF at thesame time point improved the test performance 24 h after training.

The present findings also showed that the infusion of BDNF activates in vivoERK1/2 and the transcription factor CREB, a set of cellular events obligatory toLTM formation in the CA1 region of the dorsal hippocampus (Bernabeu et al., 1997;Bourtchuladze el al., 1994; Silva et al., 1998; Taubenfeld et al., 1999). Importantly, theinfusion of anti-BDNF antibody, which blocks endogenous BDNF function, reducedERK2 and CREB activation levels. These data suggest that physiological release ofBDNF, during basal neuronal activity, may be required to maintain in vivo controllevels of activated ERK2 and CREB in the CA1 region.

Therefore, memory modulation by endogenous BDNF in the CA1 region isassociated with the regulation of ERK1/2/CREB signaling pathway. ERK1/2 andCREB have emerged as critical points of convergence in the signaling pathwaysregulating gene expression in late LTP and LTM formation (Atkins et al., 1998;Cammarota et al., 2000; Davis and Squire, 2000; Impey et al., 1998; Sweatt, 2001).

To further support this assumption, the infusion of the MEK-inhibitor PD098059reduced not only activated ERK1/2 levels but also pCREB levels in nuclear samples,endorsing the hypothesis that BDNF/ERK1/2 signaling cascade regulates pCREBlevels in vivo in hippocampal CA1 neurons.

In well agreement with our present findings, Ying et al. (2002) recently showedthat the intrahippocampal microinfusion of BDNF into the dentate gyrus triggeredLTP associated with rapid phosphorylation of ERK1/2. Activation of ERK was fur-ther coupled to phosphorylation of CREB leading to new gene expression.

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Although multiple evidence involved hippocampal cAMP/PKA cascade activa-tion in LTM formation (Bernabeu et al., 1997; Bevilaqua et al., 1997; Vianna et al.,1999) no changes were found on αPKA and βPKA catalytic subunits in nuclear sam-ples after BDNF and anti-BDNF infusion. Moreover, activated-p38, activated-PKCisoforms, and activated-AKT were unaltered after BDNF or anti-BDNF infusion.However, our results do not rule out a delayed modulation of PKA, p38, PKC,AKT, and/or CAMKII signalling pathways after BDNF or anti-BDNF infusion. Inaddition, further experiments are needed to evaluate other ERK1/2-independentsignaling pathways, like CAMK IV, possibly involved in BDNF-LTM modulation inthe hippocampus.

Taking together, our results suggest that BDNF exerts its role in LTM formationin vivo in area CA1 of the hippocampus, in a time-dependent manner, via CREBactivation. In addition, BDNF-induced CREB activation appears to be mediatedmainly through the activation of ERK1/2 signaling pathway.

ACKNOWLEDGMENTS

This work was supported by research grants from the National Research Coun-cil of Brazil (CNPq) through the National Programme for Nuclei of Excellence(PRONEX), and CONICET, ANPCyT, and the Ministry of Health of Argentina.

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Signaling mechanisms mediating BDNF modulation of memory formation in vivo in the hippocampus

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