Elevation of nerve growth factor and antisense knockdown of TrkA receptor during contextual memory consolidation

Abstract

We report here a series of experiments establishing a role for nerve growth factor and its high-affinity receptor TrkA in contextual memory consolidation. In all experiments, we trained rats in a novel chamber using tone and shock. Our first experiment revealed that endogenous nerve growth factor (NGF) increases in the hippocampus at a critical time during consolidation that occurs 1 week after training. NGF levels at other intervals (24 hr and 2 and 4 weeks after training) did not differ from those of naive control animals. In our second experiment, we blocked effects that NGF has at 1 week after training by infusing antisense TrkA phosphorothioate DNA oligonucleotide. Reduction of septohippocampal TrkA receptor expression selectively impaired memory consolidation for context but not for tone. Animals with antisense TrkA oligonucleotide infused into the medial septal area or CA1 of the hippocampus froze less when placed in the training chamber than did animals infused with inactive randomized oligonucleotide. At 4 weeks after training, antisense TrkA oligonucleotide had no effect on freezing. Third, we correlated levels of freezing with choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) immunohistochemistry. Antisense TrkA infused into CA1 of the hippocampus reduced cell body cross-sectional area for cholinergic cells in the medial septal area and decreased the density of hippocampal terminals labeled for ChAT and VAChT proteins. Cholinergic cell body measurements were significantly correlated with freezing. Taken together, these results indicate a role for nerve growth factor acting via the TrkA receptor on ChAT and VAChT proteins in contextual memory consolidation.

Fig. 1.

Schematic diagram of a typical infusion site of antisense TrkA oligonucleotide into CA1 of the dorsal hippocampus. The extent was deduced from reduced immunohistochemical staining for ChAT and VAChT. The template is from Paxinos and Watson (1997). CA1, CA2,CA3, Ammon's horn, regions 1–3; DG, dentate gyrus.

Fig. 2.

Behavioral responses at different training–testing intervals. A, The percentage of time animals were freezing (immobile except for respiratory movements) when placed in the training chamber. Values are for naive control animals and animals with 0 (24 hr), 1, 2, and 4 week training–testing intervals (4 animals in each group). Error bars show the SEM.Asterisks denote behavioral response levels that differed significantly from that of naive controls (p < 0.01). B, NGF levels in hippocampal homogenates collected from both sides of the brains of the naive control animals and the animals killed at 0 (24 hr), 1, 2, and 4 week intervals after training. Error bars show the SEM. At 1 week after training (see asterisk), NGF levels were significantly higher (p < 0.05) compared with that of naive control animals that were tested but not trained.

Fig. 3.

Behavioral outcomes of antisense oligonucleotide experiments. A, Freezing to the chamber is significantly decreased (asterisk, p < 0.05) by unilateral infusions of antisense TrkA oligonucleotide into the medial septal area as compared with unilateral infusions of randomized control oligonucleotide. This was the outcome 1 week after training and 24 hr after surgery. B, Freezing to the chamber is significantly decreased (asterisk, p< 0.01) by bilateral infusions of antisense TrkA oligonucleotide into the medial septal area as compared with bilateral infusions of randomized control oligonucleotide. This was the outcome 1 week after training and 24 hr after surgery. C, Freezing to the chamber is significantly decreased (asterisk,p < 0.05) by bilateral infusions of antisense TrkA oligonucleotide into CA1 of the hippocampus as compared with bilateral infusions of randomized control oligonucleotide. This was the outcome 1 week after training and 48 hr after surgery. D, Freezing to the chamber is not affected by bilateral infusions of antisense TrkA oligonucleotide into the medial septal area as compared with bilateral infusions of randomized control oligonucleotide 4 weeks after training and 24 hr after surgery. Error bars in A–D show the SEM; 3–4 animals served as subjects in each group of the above experiments.

Fig. 4.

Loss of cholinergic markers after antisense TrkA oligonucleotide infusions into the medial septal area. A, B, Brain sections through the medial septal area were immunohistochemically processed for ChAT. A, An animal given randomized control oligonucleotide is shown. B, An animal given antisense TrkA oligonucleotide is shown. C, D, Brain sections through the medial septal area were immunohistochemically processed for VAChT. C, An animal given randomized control oligonucleotide is shown. D, An animal given antisense TrkA oligonucleotide is shown. E, F, Brain sections through the medial septal area were immunohistochemically processed for TrkA receptor. E, An animal given randomized control oligonucleotide is shown.F, An animal given antisense TrkA oligonucleotide is shown. ms, Medial septal nucleus; vdb, vertical diagonal band nucleus. Scale bar: A–F, 300 μm.

Fig. 5.

Terminals in CA1 of the hippocampus. VAChT-immunopositive terminals in CA1 of the hippocampus are intact after local infusions of randomized control oligonucleotide (A) but are substantially reduced in the number in rats receiving antisense TrkA oligonucleotide (B). Synaptophysin-immunopositive terminals in CA1 of the hippocampus after local infusions of randomized control oligonucleotide (C) or antisense TrkA oligonucleotide (D) remain equally intact. Scale bar: A–D, 20 μm.

Fig. 6.

Remote effects on cholinergic cells. ChAT-immunopositive cells in the medial septal area have unaltered cell bodies after local infusions of randomized control oligonucleotide (A, C), but cells are visibly reduced in cross-sectional area after receiving antisense TrkA oligonucleotide (B, D). ms, Medial septal nucleus;vdb, vertical diagonal band nucleus. Scale bar:A, B, 400 μm (low power); C, D, 40 μm (high power).

Fig. 7.

Graphs depicting correlations between cell body cross-sectional area versus percentage of time freezing. A total of 1498 cells were measured. Black triangles represent the average cell body cross-sectional area for animals receiving antisense TrkA oligonucleotide, and white triangles represent the average cell body cross-sectional area for animals receiving randomized control oligonucleotide. Linear plots illustrate the relationships between ChAT-immunopositive (A) or VAChT-immunopositive (B) cells and behavioral responses. Correlations were significant for both ChAT-immunopositive (p < 0.005) and VAChT-immunopositive (p < 0.05) cell body cross-sectional areas and percentage of time freezing.