N-methyl-D-aspartate-stimulated ERK1/2 signaling and the transcriptional up-regulation of plasticity-related genes are developmentally regulated following in vitro neuronal maturation

Abstract

The general features of neuroplasticity are developmentally regulated. Although it has been hypothesized that the loss of plasticity in mature neurons may be due to synaptic saturation and functional reduction of N-methyl-D-aspartate receptors (NMDAR), the molecular mechanisms remain largely unknown. We examined the effects of NMDAR activation and KCl-mediated membrane depolarization on ERK1/2 signaling following in vitro maturation of cultured cortical neurons. Although NMDA stimulated a robust increase in intracellular calcium at both DIV (day in vitro) 3 and 14, the activation of ERK1/2 and cAMP responsive element-binding protein (CREB) was impaired at DIV 14. Specifically, the phosphorylation of ERK1/2 was stimulated by both NMDA and KCl at DIV 3. However, at DIV 14, NMDA- but not KCl-stimulated ERK1/2 and CREB phosphorylation was significantly diminished. Consistently, the NMDA-induced transcription of ERK/CREB-regulated genes Bdnf exon 4, Arc, and zif268 was significantly attenuated at DIV 14. Moreover, in comparison with 3 DIV neurons, the phosphorylated-ERK1/2 in 14 DIV neurons displayed a tremendous increase following maturation and was more susceptible to dephosphorylation. Blocking calcium channels by nifedipine or NMDAR by APV caused a more dramatic ERK dephosphorylation in 14 DIV neurons. We further demonstrate that the loss of plasticity-related signaling is unrelated to NMDA-induced cell death of the 14 DIV neurons. Taken together, these results suggest that the attenuation of certain aspects of neuroplasticity following maturation may be due to the reduction of NMDAR-mediated gene transcription and a saturation of ERK1/2 activity.

Fig. 1

The activation of ERK1/2 and CREB declines following in vitro maturation. Cortical neurons were stimulated by 50 mM KCl (a, b, and c) or 50 uM NMDA2 uM glycine (b and c) at DIV3 and 14. (a). KCl stimulated ERK1/2 phosphorylation at both DIV 3 and 14. For (b) and (c), neurons were pretreated with CNQX and nifedipine, or CNQX and APV, as indicated, for 30min before the stimulation by KCl (along with APV and CNQX) and NMDA/glycine (along with nifedipine and CNQX). Samples were collected 15 min after stimulation. The signal of p-ERK1/2 was normalized with total ERK1/2 or beta-actin. The signal of p-CREB was normalized with beta-actin. Data are average +/− SD (n=5 for each group).

Fig. 2

Time course of ERK1/2 and CREB activation. DIV 3 (a and c) and 14 (b and d) cortical neurons were stimulated by NMDA or KCl as indicated. The samples were collected 5 min, 15 min, 60 min, and 120 min after stimulation, and the level of p-ERK1/2 and p-CREB was analyzed by Western blots. For (a) and (b), the neurons were pre-treated with CNQX and nifedipine for 30min before the application of NMDA/glycine (along with CNQX and nifedipine). For (c) and (d), neurons were pre-treated with CNQX and APV for 30 min before the application of KCl (along with CNQX and APV). The levels of p-ERK1/2 and p-CREB were normalized to beta-actin. The representative Western blots are shown in the upper panels, and the quantification in the lower panels. Data are average +/− SD (n=5 for each group).

Fig. 3

NMDA-stimulated transcription of Arc, zif268, and Bdnf exon 4 declines following maturation. DIV 3 (a and c) and 14 (b and d) cortical neurons were stimulated by 50 mM KCl or 50 uM NMDA/2 uM glycine, as indicated. The samples were collected 30 min, 60 min, and 120 min after stimulation, and the mRNA levels of Arc, zif268, and Bdnf exon 4 were analyzed by semi-quantitative RT-PCR. For (a) and (b), the neurons were pre-treated with CNQX and nifedipine for 30 min before the application of NMDA/glycine (along with CNQX and nifedipine). For (c) and (d), neurons were pre-treated with CNQX and APV for 30 min before the application of KCl (along with CNQX and APV). Similar results were obtained from 4 independent experiments.

Fig. 4

Membrane depolarization and NMDAR activation induce significant calcium influx in mature neurons. The intracellular Ca²⁺ concentration, [Ca²⁺]_i, was measured by the Fura ratio (F340/380) in cortical neurons at DIV 3 and 14. For (a) and (b), baseline level of [Ca²⁺]_i was obtained when perfused with PPS, then the neurons were perfused with nifedipine and CNQX in PPS for 10min. The peak level of [Ca²⁺]_i was measured 5 min after NMDA (50 uM)/glycine (2 uM) (in PPS containing nifedipine and CNQX) stimulation. For (c) and (d), neurons were perfused with APV and CNQX in PPS for 10 min. The peak value of [Ca²⁺]_i was measured 5 min after KCl (in PPS containing APV and CNQX) stimulation. The ratio of F340/380 was arbitrarily set to 1 for the untreated control neurons, and the relative changes in F340/380 ratio with stimulated neurons was normalized to the control level. Quantifications are shown in (a) and (c) (expressed as average +/− SD). Pseudo-colored images of neurons before (control) and after treatments (as indicated) are shown in (b) and (d). The absolute ratios of F340/380 for untreated control neurons at DIV 3 and 14 are shown in (e) (expressed as average +/− SD).

Fig. 5

Total expression and the phosphorylation of ERK1/2 are significantly up-regulated following in vitro maturation. Primary cortical cultures were examined for the level of p-ERK1/2 and total ERK1/2 at DIV 3, 6 and 14. Representative Western blots are shown in (a). The relative levels of total ERK1/2 (T-ERK1/2) were normalized to beta-actin and quantified (n=5 from each group) (b). To obtain the profile for ERK1/2 activation during the development, the level of p-ERK1/2 was normalized with T-ERK1/2 (c). The relative expression level at DIV 3 was arbitrarily set as 1. Data are average ± S.D. (d) and (e), DIV 14 neurons show more dephosphorylation of p-ERK1/2 when L-VGCC and NMDAR are blocked. Samples were collected from the control and nifedipine/CNQX- or APV/CNQX-treated neurons, and analyzed for the level of p-ERK1/2. The representative Western blots are shown in the upper panels, and quantification in the lower panels (average +/− SD, n=4). (f) and (g), traces of the F340/380 ratios (measured once every 5 sec) show [Ca²⁺]_i levels in DIV 3 (f) and 14 (g) cortical neurons. Traces were selected randomly for 3 neurons. The duration of nifedipine/CNQX treatment is shown as indicated.

Fig. 6

Bath incubation with NMDA at 10 uM does not cause significant excitotoxic cell death. The neuronal cell death was triggered by NMDA as described in Materials and Methods. DAPI staining demonstrated that NMDA (at 10 uM and 50 uM) did not induce any apparent cell death in DIV 3 neurons. Although high concentration of NMDA (at 50 uM) induced significant cell death in DIV 14 neurons (*: p<0.05), low concentration of NMDA (at 10 uM) did not. Representative DAPI staining is shown in A, and quantification (from 6 independent cultures for both DIV 3 and DIV 14) is shown in B.

Fig. 7

The NMDA-stimulated plasticity-related signaling is significantly dampened following maturation. (a) The non-toxic concentration of NMDA (at 10 uM) stimulated significant more phosphorylation of ERK and CREB at DIV 3 than at DIV 14. Neurons were pre-treated with CNQX and nifedipine for 30 min, then stimulated with NMDA/glycine (in the presence of CNQX and nifedipine). The samples were harvested 15 min after stimulation, and analyzed by Western blots (n=4; *: p<0.05). (b) DIV 3 and DIV 14 neurons were treated as described in (a). The samples were collected 1 hr after the stimulation, the mRNA level of zif268, Arc, and Bdnf exon 4 was examined by semi-quantitative RT-PCR (n=4; *: p<0.05).

Fig. 8

The transcriptional up-regulation of zif268, Arc, and bdnf exon 4 requires prolonged synaptic activation by bicuculline. DIV 3 or 14 neurons were treated with bicuculline (50 uM) and analyzed for p-ERK and p-CREB (a) 30 min after stimulation (n=3). For b and c, the transcription of zif268, Arc, and Bdnf exon 4 was analyzed by RT-PCR. Samples were collect 1 hr (b) or 4 hrs (c) after bicuculline stimulation (n=4). RT-PCR did not reveal any transcriptional up-regulation 1 hr after bicuculline stimulation. The representative Western blots or RT-PCR results are displayed in the left panels. The quantifications are shown in the right panels (average +/1 SD; *:p<0.05). The development stages (DIV 3 and DIV 14) and treatments are indicated in the figure.

Fig. 9

Co-application of okadaic acid and NMDA results in the activation of ERK, CREB, as well as the transcription of zif268, Arc, and Bdnf exon 4 in DIV 14 neurons. DIV 14 cortical neurons were pre-treated with a general protein phosphatase inhibitor okadaic acid (1uM), or PP2A inhibitor cantharidin (0.1uM), or PP2B inhibitor FK506 (1uM) for 30 min before NMDA (50 uM)/glycine (2 uM) stimulation. (a). To determine the level of p-ERK1/2 and p-CREB, samples were harvested 15 min after NMDA (50 uM)/glycine/nifedipine/CNQX stimulation, and analyzed by Western blots. (b). To determine the mRNA level of Arc, zif268 and Bdnf exon 4, samples were collected 60 min after NMDA (50 uM)/glycine/nifedipine/CNQX application. A 30 min pre-treatment with nifedipine and CNQX was included for all samples. Representative images are shown in the upper panels, and quantification in the lower panels (average +/− SD; n=4). The level of RT-PCR products of the individual genes, p-ERK, and p-CREB in the control neurons was set as 1.