Antisense oligodeoxynucleotide-mediated disruption of hippocampal cAMP response element binding protein levels impairs consolidation of memory for water maze training

Abstract

Extensive evidence suggests that long term memory (LTM) formation is dependent on the activation of neuronal second messenger systems and requires protein synthesis. The cAMP response element binding protein (CREB) is a constitutively expressed regulatory transcription factor that couples changes in second messenger levels to changes in cellular transcription. Several recent studies suggest that CREB and related transcription factors regulate gene expression necessary for neuronal plasticity and LTM. However, the role of CREB, within defined mammalian brain structures, in mediating the cellular events underlying LTM formation has not been investigated. We examined whether CREB-mediated transcription within the dorsal hippocampus is critical to LTM consolidation of water maze spatial training, which is known to depend on dorsal hippocampal function. Pretraining infusions of antisense oligodeoxynucleotides (ODN) directed against CREB mRNA were used to disrupt hippocampal CREB protein levels in adult rats. Control groups received pretraining infusions of ODN of the same base composition but in a randomized order (scrambled ODN) or buffer. Task acquisition and memory up to 4 h (i.e., short term memory) were similar in CREB antisense ODN and control groups. In contrast, CREB antisense ODN-infused rats exhibited significantly impaired memory 48 h later (i.e., LTM). Moreover, administration of antisense ODN 1 day after training did not affect subsequent retention performance. These findings provide the first evidence that CREB-mediated transcription is integral to hippocampal-dependent memory consolidation processes.

Figure 1

Biotinylated S-ODN in the dorsal hippocampus: Distribution and relative levels at 2 and 20 h postinfusion. (A) CREB antisense 20-mer S-ODN (1 nmol in 1 μl) with an added 5′ biotin group was infused into the left hippocampus while the same amount of unlabeled S-ODN was infused into the right hippocampus. Rats were killed 3 h later. Biotinylated S-ODN is indicated by dark staining. Three sections from a representative rat are shown, demonstrating the rostral–caudal extent of S-ODN diffusion. (B) CREB antisense biotinylated S-ODN (2 nmol in 1 μl) were infused unilaterally (left). Rats were killed 2 or 20 h later, followed by detection of biotinylated S-ODN. Representative brains from each time point are shown.

Figure 4

Twenty-h pretreatment with CREB antisense S-ODN impairs 48-h retention performance, but not acquisition, of the water maze task. (A) CREB antisense or scrambled S-ODN were administered bilaterally ≈20 h before training (2 nmol in 1 μl; n = 14–15 rats/group). A retention test consisting of three trials was given 2 days later. No significant differences were seen for each of the 10 individual training trials or for the training sessions as a whole (P > 0.05). The CREB antisense group performed significantly worse on the retention test as a whole (F_1,27 = 14.4; ∗∗, P < 0.001) and on retention trials 1 and 2 (F_1,27 = 8.6 for trial 1 and F_1,27 = 8.0 for trial 2; ∗, P < 0.01) compared with the scrambled control group. (B) Naive cannulated rats were trained as described above. The following day, rats were counterbalanced into two groups based on training performance and then given bilateral infusions of either CREB antisense or scrambled S-ODN (2 nmol in 1 μl; n = eight rats/group). Retention was tested 3 days later; this delay matches the time interval between S-ODN infusions and retention testing as in the experiment shown in A. No significant differences were observed in the retention test as a whole or in any of the individual retention trials (P > 0.05).

Figure 3

Six-hour pretreatment with CREB antisense EC-ODN impairs 48-h retention performance, but not acquisition, of the water maze task. (A) PBS, CREB antisense EC-ODN, or scrambled EC-ODN were administered bilaterally 6 h before training in the water maze (2 nmol in 1 μl; n = 9–12 rats/group). A retention test consisting of three trials was given 2 days later. No significant differences were seen for each of the 12 individual training trials or for the training sessions as a whole (P > 0.05). The CREB antisense group performed significantly worse on the retention test as a whole (F_2,30 = 5.8; P < 0.01; CREB antisense vs. scrambled, ∗∗, P < 0.005; CREB antisense vs. PBS, ∗, P < 0.02; scrambled vs. PBS, P = 0.90) and retention trial 2 compared with both control groups (F_2,30 = 5.5; P < 0.01; CREB antisense vs. scrambled or PBS, #, P < 0.01; scrambled vs. PBS, P = 0.77). (B) Swim path lengths from the retention session shown in A were analyzed. The CREB antisense group swam significantly longer path lengths in the retention test as a whole than either control group (F_2,30 = 6.6; P < 0.005; CREB antisense vs. scrambled, ∗∗, P < 0.005; CREB antisense vs. PBS, ∗, P < 0.01; scrambled vs. PBS, P = 0.82). The CREB antisense ODN-treated rats also swam significantly longer path lengths on retention trials 1 and 2 than either control group (retention trial 1: F_2,30 = 3.7; P < 0.05; CREB antisense vs. scrambled or PBS, #, P < 0.05; PBS vs. scrambled, P = 0.99; retention trial 2: F_2,30 = 6.6; P < 0.005; CREB antisense vs. scrambled or PBS, ##, P < 0.005; PBS vs. scrambled, P = 0.81).

Figure 2

CREB levels in the dorsal hippocampus are specifically decreased 6 h after in vivo antisense ODN treatment. Rats received infusions of CREB antisense ODN in one hippocampus and scrambled ODN in the other before being killed and before tissue dissection. Three different treatment conditions were included: treatment 1, rats received EC-ODN infusions and were killed 6 h later; treatment 2, rats received S-ODN infusions and were killed 6 h later; treatment 3, rats received EC-ODN infusions and were killed 54 h later. In all cases, 2 nmol of ODN (in 1 μl) was infused; antisense ODN infusions were alternated between left and right hippocampi. Tissue punches were taken from dorsal and ventral hippocampi, and protein extracts were prepared. Extracts for each treatment, group, and punch location (i.e., treatment 1, group 1, CREB antisense ODN, dorsal hippocampus) were pooled for immunoblot analysis. Group pools were obtained from three to five rats; the number of groups given each treatment condition is noted. Immunoblot analysis was performed sequentially for CREB and then GluR1. Levels of CREB immunoreactivity were normalized to those of GluR1 for each sample. Additionally, treatment 1 dorsal hippocampal extracts were analyzed for ATF-2 immunoreactivity; as for CREB, ATF-2 levels were normalized to GluR1. Values were obtained from two to three independent blots. Normalized CREB levels from CREB antisense ODN samples are expressed as a percentage of normalized CREB levels from scrambled ODN samples for the same group. CREB antisense EC-ODN infused into the dorsal hippocampus 6 h before death produced a specific, significant reduction in CREB (∗∗, P < 0.02, t test) but not in ATF-2.