Brain-derived neurotrophic factor induces long-term potentiation in intact adult hippocampus: requirement for ERK activation coupled to CREB and upregulation of Arc synthesis

Abstract

Brain-derived neurotrophic factor (BDNF) is implicated in long-term synaptic plasticity in the adult hippocampus, but the cellular mechanisms are little understood. Here we used intrahippocampal microinfusion of BDNF to trigger long-term potentiation (BDNF-LTP) at medial perforant path--granule cell synapses in vivo. BDNF infusion led to rapid phosphorylation of the mitogen-activated protein (MAP) kinases ERK (extracellular signal-regulated protein kinase) and p38 but not JNK (c-Jun N-terminal protein kinase). These effects were restricted to the infused dentate gyrus; no changes were observed in microdissected CA3 and CA1 regions. Local infusion of MEK (MAP kinase kinase) inhibitors (PD98059 and U0126) during BDNF delivery abolished BDNF-LTP and the associated ERK activation. Application of MEK inhibitor during established BDNF-LTP had no effect. Activation of MEK-ERK is therefore required for the induction, but not the maintenance, of BDNF-LTP. BDNF-LTP was further coupled to ERK-dependent phosphorylation of the transcription factor cAMP response element-binding protein. Finally, we investigated the expression of two immediate early genes, activity-regulated cytoskeleton-associated protein (Arc) and Zif268, both of which are required for generation of late, mRNA synthesis-dependent LTP. BDNF infusion resulted in selective upregulation of mRNA and protein for Arc. In situ hybridization showed that Arc transcripts are rapidly and extensively delivered to granule cell dendrites. U0126 blocked Arc upregulation in parallel with BDNF-LTP. The results support a model in which BDNF triggers long-lasting synaptic strengthening through MEK-ERK and selective induction of the dendritic mRNA species Arc.

Fig. 1.

MEK inhibitors block BDNF-LTP. Time course plots of medial perforant path-evoked fEPSP and population (Pop) spike responses. Test pulses were applied at a rate of 1 every 30 sec. BDNF (2 μg/2 μl) was infused 300 μm above the medial perforant–granule cell synapses, during the period indicated by the white bar. n = 16.a, Effect of PD98059 on BDNF-LTP. Infusion of 1 μl of PD98059 (30 μm) was followed immediately by infusion of 2 μg of BDNF in 2 μl of PD98059. The period of MEK inhibitor (black bar) and BDNF plus MEK inhibitor (hatched bar) application are indicated. Values are means ± SEM expressed in percentage of baseline. n = 8.Inset above shows traces of averaged field responses (5 sweeps) recorded at the time points indicated. b, Effect of U0126 (30 μm) on BDNF-LTP in a separate groups of animals. n = 8. BDNF-LTP development is abolished by both MEK inhibitors. The group receiving BDNF alone is the same ina and b. c, Cytochromec (2 μg/2 μl; n = 8) had no significant effect on baseline transmission. d, PD98059 (30 μm; n = 6) and U0126 (30 μm; n = 4) applied alone had no effect on baseline synaptic transmission. fEPSP slope data are shown.Insets are averaged traces as done in c. Infusion of PBS–DMSO vehicle also had no effect on baseline transmission (n = 4; data not shown). Calibration: 3 mV, 4 msec.

Fig. 2.

Effect of the MEK inhibitor U0126 on established BDNF-LTP. a, U0126 (30 μm, 2 μl) was infused 110 min after BDNF infusion, during established BDNF-LTP. MEK inhibitor treatment had no significant effect on medial perforant path-evoked responses during infusion or during the subsequent 2 hr recording period. n = 6. b, Effect of vehicle (DMSO–PBS) infusion on established BDNF-LTP.n = 6. c, Summary bar graph of fEPSP slope increases obtained in groups receiving BDNF alone or BDNF and post-treatment with U0126 or PBS. Values are group means + SEM) based on 20 responses collected at the end of recording, 245–255 min after BDNF infusion. n = 6 in all groups. There was no significant difference between groups in the magnitude of BDNF-LTP (p > 0.05).

Fig. 3.

BDNF-LTP is coupled to enhanced ERK phosphorylation in the dentate gyrus. Western blot assays of phosphorylated, active ERK (p-ERK1/2) were run on aliquoted samples from microdissected dentate gyrus (DG) and hippocampal regions CA1 and CA3 after in vivo electrophysiological experiments. a, Group mean + SEM changes in p-ERK2 immunoreactivity levels based on densitometric analysis. Optical density values are expressed as a ratio between the treated and nontreated (control) side for each hippocampal subfield. BDNF infusion increased activation of ERK2 at 15 min (n = 8) and 3 hr (n = 7) in the infused dentate gyrus. BDNF had no effect on ERK2 phosphorylation in hippocampal region CA1 or CA3. Delivery of the MEK inhibitor U0126 abolished the increase in ERK2 phosphorylation at both 15 min and 3 hr. n = 4 at both time points. Cytochrome c (Cyt C) infusion had no effect on p-ERK2 immunoreactivity levels. *p < 0.05, significant difference from control.b, Representative p-ERK immunoblots for the group data shown in a. Total ERK2 protein levels were unchanged.c, Bar graph of the fEPSP slope changes obtained at 15 min and 3 hr after infusion. *p < 0.05, significant difference from baseline.

Fig. 4.

BDNF-LTP is associated with enhanced activation of p38 MAPK but not JNK. a, Group mean + SEM changes in phosphorylated kinase immunoreactivity levels based on densitometric analysis. Optical density values are expressed as a ratio between the treated and nontreated (control) dentate gyrus. BDNF infusion increased phospho-p38 immunoreactivity at 15 min (n = 8) and 3 hr (n = 7) in the infused dentate gyrus. BDNF infusion had no effect on JNK phosphorylation. Cytochromec (Cyt C) infusion had no effect on p-p38 or p-JNK immunoreactivity levels. There were no changes in phosphorylation of these kinases in region CA1 or CA3 (data not shown). Total protein levels of p38 and JNK were unchanged in all hippocampal regions across treatments. *p < 0.05, significant difference from control. b, Representative Western blots for group data shown in a.

Fig. 5.

BDNF-LTP is coupled to rapid CREB phosphorylation.a, Group mean + SEM changes in p-CREB immunoreactivity levels based on densitometric analysis. CREB-Ser¹³³was rapidly phosphorylated at 15 min (n = 8), returning to control levels at 3 hr (n = 7) after BDNF infusion. Infusion of the U0126 blocked CREB activation. *p < 0.05, significant difference from control.b, p-CREB Western blots for group data ina.

Fig. 6.

BDNF-LTP is coupled to upregulation of Arc mRNA and protein. a, Group mean + SEM changes inArc and Zif268 protein immunoreactivity levels.Arc protein expression was increased at 3 hr (n = 7), but not 15 min (n = 8), after BDNF infusion. These increases were confined to the infused dentate gyrus. U0126 blocked the increases in Arcexpression. BDNF infusion had no effect on Zif268 protein expression. *p < 0.05, significant difference from control.b, Representative Western blots for group data ina. c, Autoradiographs showing in situ hybridization signals for Arc andZif268 in the hippocampus 2 hr after BDNF or cytochromec infusion into the left dentate gyrus. Note the robust increase in the hybridization signal for Arc mRNA in the treated dentate gyrus. d, High-magnification autoradiographic image of the Arc mRNA signal in the dentate gyrus. Model granule cells with apical dendrites extending throughout the molecular layer are depicted in the top and bottom blades of the dentate gyrus. Arc mRNA is strongly upregulated in granule cell bodies and extensively distributed in granule cells dendrites.

Fig. 7.

Model of BDNF signaling pathways underlying BDNF-LTP in the dentate gyrus in vivo. Exogenous application of BDNF triggers LTP through activation of MEK-ERK. ERK activation is required for both CREB phosphorylation and induction of the immediate early gene Arc in dentate granule cells.Arc and Zif268 are both required in late HFS-LTP (Guzowski et al., 2000; Jones et al., 2001), yet they are likely to mediate distinct components of the process;Arc is rapidly delivered to dendrites and translated, whereas Zif268 regulates expression of delayed response genes. Our data suggests the hypothesis that BDNF triggers late LTP through ERK-dependent induction of Arc. HFS must recruit additional pathways to induce Zif268 expression, leading to transcription of late effector genes and full expression of late LTP.