Article Figures & Data
Figures
- Fig. 1.
Experience-induced increase in α-CaMKII protein in the visual cortex mediated by NMDAR activation and mRNA polyadenylation. A, Quantification of α-CaMKII levels in synaptoneurosome (SN) fractions isolated from the visual cortex of animals reared in complete darkness (DR) and animals reared in the dark and exposed to light for 30 min (DR + 30′). Western blots for α-CaMKII and NMDAR subunit NR1 were performed from PAGE loaded with equal total protein of SN samples isolated from DR and DR + 30′ visual cortex. Quantitative densitometry was performed on the α-CaMKII bands, and these were normalized to the level of NR1 in the same lane [the amount of NR1 subunit in SN fraction does not change with visual experience (Quinlan et al., 1999)]. Where indicated, actinomycin D (1 mg/kg) was injected (intraperitoneally) 30 min before light exposure. This dose of actinomycin D is effective in blocking protein synthesis in the brain (Jackson, 1972; Pickering and Fink, 1976). Each experiment consisted of two to four rats per treatment group, and results shown are the mean ± SEM of three experiments. Insets show representative bands from one experiment. B, Quantification of α-CaMKII expression as in A, in animals injected with the NMDAR antagonist CPP (10 mg/kg) 30 min before light exposure. Each experiment consisted of two to four rats per treatment group, and results shown are the mean ± SEM of three experiments. C, Quantification of α-CaMKII performed as in A, in animals injected with cordycepin (6 mg/kg) 30 min before light exposure. Each experiment consisted of two to four rats per treatment group, and results shown are the mean ± SEM of three experiments.
- Fig. 2.
Transfection of hippocampal cells in culture with reporter GFP constructs. A, Schematic of α-CaMKII mRNA and the GFP constructs used for transfections. GFP constructs were modified to contain the last ∼160 nucleotides of α-CaMKII 3′-UTR with either intact CPE sequences (GFP-CPEWT;top) or mutated CPEs (GFP-CPEMUT;bottom). B, Hippocampal neurons grown in culture for 7 d, transfected with GFP-CPEWT. This culture was processed for GFP fluorescence 8 hr after transfection. GFP-fluorescing neurons are readily distinguished from non-GFP-fluorescing neurons, and GFP is detected throughout the entire neuron (right). Scale bar, 20 μm.
- Fig. 3.
GFP mRNA and GFP protein expression in transfected neurons. Neurons were processed for GFP fluorescence (GFP-Fl) and fluorescent in situhybridization (GFP-ISH) at either 6 or 24 hr after transfection. Neurons at 6 hr can contain GFP mRNA without expressing detectable GFP fluorescence (top panel). In contrast, at 24 hr after transfection, all GFP mRNA-containing neurons also express the fluorescent GFP protein (middle panel). In the bottom panel, the anti-DIG primary antibody was replaced with a nonspecific normal mouse IgG antibody (control IgG). DAPI staining reveals the nuclei of cells within each field.
- Fig. 4.
Quantification of GFP expression in cultured hippocampal neurons and experimental design. A, GFP fluorescence is not correlated with GFP mRNA expression at early times after transfection. In cultures transfected with GFP-CPEWT, 9.23 ± 0.002% of all neurons expressed GFP mRNA at 6 hr. However, significantly fewer neurons (3.4 ± 0.002%) expressed GFP protein at detectable levels (p ≤ 0.005). In contrast, at 24 hr after transfection, 10.27 ± 0.003% of the total neuronal population contained GFP mRNA, and 9.41 ± 0.004% exhibited GFP fluorescence (p = 0.09). Similar results were obtained when cultures were transfected with GFP-CPEMUTconstructs: at 6 hr after transfection, 9.3 ± 0.002% contained GFP mRNA, with only 3.07 ± 0.002% expressing detectable protein (p ≤ 0.01). This difference was not present at 24 hr after transfection (10.2 ± 0.002% contained GFP mRNA, and 9.7 ± 0.005% contained GFP fluorescence;p = 0.4). Data represent mean ± SEM.B, Experimental design. Seven- to 10-d-old cultures were transfected and then stimulated with either glutamate or KCl at time points between 4.5 and 24 hr after transfection. GFP fluorescence and GFP mRNA presence (using fluorescent in situhybridization) was scored 1.5 hr after stimulation.GFP-Fl, GFP fluorescence; GFP-ISH, GFP-fluorescent in situ hybridization.
- Fig. 5.
Activity induces an increase in translation of CPE-containing mRNA at early times after transfection.A, Hippocampal neuron cultures transfected with either GFP-CPEWT (black bars) or GFP-CPEMUT (gray bars) were stimulated 6 hr after transfection by a 30 sec application of glutamate (glu; 100 μm) and processed for GFP fluorescence 1.5 hr later. A significant increase in the number of GFP-expressing neurons was detected only in the cultures transfected with the CPE-containing construct (n = 3).B, The glutamate-induced increase in GFP-expressing neurons is dependent on protein synthesis. Cultures transfected with GFP-CPEWT and stimulated with glutamate (glu) as above were treated with cycloheximide 30 min before glutamate stimulation. Cycloheximide (cyc) treatment blocked the increase in the number of GFP-expressing neurons. Cycloheximide treatment alone for the duration of the post-stimulation period (1.5 hr) had no effect on the number of GFP-expressing neurons (n = 3). con, Control.C, Depolarization induces an increase in GFP-expressing neurons that is not dependent on new gene transcription. Where indicated, cultures transfected with GFP-CPEWT were depolarized with KCl (35 mm, 5 min) 1.5 hr before fixation. KCl depolarization induced a significant increase in the number of GFP-expressing neurons. Addition of actinomycin D (Act. D; 25 μm) 30 min before KCl application did not alter the response to KCl depolarization (n = 3).D, Time course of GFP expression in neurons transfected with GFP-CPEWT. Hippocampal neurons were transfected with GFP-CPEWT and then processed for GFP fluorescence at 6, 8, 10, and 14 hr after transfection (♦). In parallel experiments, cultures were stimulated with 35 mmKCl for 5 min (arrows at 4.5, 6.5, 8.5, and 10.5 hr) 1.5 hr before fixation (▪). Three coverslips were counted at each time point in each experiment, and results are the mean ± SEM of two experiments. All coverslips were counted blind to treatment protocol (control is unstimulated; *p ≤ 0.05).
- Fig. 6.
Activity-dependent translation in cultured hippocampal neurons regulated by NMDAR activation and mediated by polyadenylation. A, Neurons cultured and transfected as in Figure 5 were treated with the NMDAR antagonist APV (300 μm) starting immediately after transfection and continuing through the end of the stimulation protocol (total of 7.5 hr). The glutamate (glu)-induced increase in the number of neurons expressing GFP in cultures transfected with GFP-CPEWT was inhibited by APV. APV treatment alone for the entire post-transfection interval (7.5 hr) caused a small but significant (p < 0.05) decrease in GFP expression.B, The KCl depolarization-induced increase in GFP-expressing neurons was similarly inhibited by APV (n = 4). C, The glutamate-induced stimulation of GFP translation is blocked by the treatment of cordycepin (cordy; 200 μm) for 30 min before glutamate stimulation (n = 3). Cordycepin alone did not affect GFP expression in these neurons (control is unstimulated; *p ≤ 0.05).
