Upregulation of polysialylated neural cell adhesion molecule in the dorsal hippocampus after contextual fear conditioning is involved in long-term memory formation

Abstract

The role of the hippocampus in pavlovian fear conditioning is controversial. Although lesion and pharmacological inactivation studies have suggested a key role for the dorsal hippocampus in contextual fear conditioning, the involvement of the ventral part is still uncertain. Likewise, the debate is open with regard to the putative implication of each hippocampal subdivision in fear conditioning to a discrete conditioned stimulus. We explored the potential existence of dissociations occurring in the dorsal versus ventral hippocampus at the cellular level while dealing with either contextual or cued fear conditioning and focused in a molecular "signature" linked to structural plasticity, the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). We found an upregulation of PSA-NCAM expression in the dorsal (but not ventral) dentate gyrus at 24 h after contextual (but not tone) fear conditioning. Specific removal of PSA through microinfusion of the enzyme endoneuraminidase-N in the dorsal (but not ventral) hippocampus reduced freezing responses to the conditioned context. Therefore, we present evidence for a specific role of PSA-NCAM in the dorsal hippocampus in the plasticity processes occurring during consolidation of the context representation after "standard" contextual fear conditioning. Interestingly, we also found that exposing animals just to the context induced an activation of PSA-NCAM in both dorsal and ventral dentate gyrus. Altogether, these findings highlighting the distinctive occurrence of these neuroplastic processes in the dorsal hippocampus during the standard contextual fear-conditioning task enlighten the ongoing debate about the involvement of these hippocampal subdivisions in pavlovian fear conditioning.

Figure 1.

Contextual, but not tone, fear conditioning increases PSA-NCAM expression in the dentate gyrus of the hippocampus. A, B, Percentage of time spent freezing during the context (A) and tone (B) tests, 30 min or 24 h after fear conditioning under two different shock intensities (0.4 or 1 mA). C, D, PSA-NCAM-labeled cells in the dentate gyrus. Representative photomicrographs of PSA-NCAM staining of the dentate gyrus in a naive rat (C) and in an animal submitted to contextual fear conditioning (1 mA shock intensity) (D). E, F, Number of polysialylated cells in the dentate gyrus 30 min or 24 h after training animals in either a contextual (E) or a tone (F) fear-conditioning paradigm. Data represent mean ± SEM. Thickest horizontal discontinuous line represents mean values of the naive group, and thinner ones represent SEM values of this group. H, Hilus. *p < 0.05, **p < 0.01, ***p < 0.001 versus naive; ^#p < 0.001 versus 1 mA group in the 30 min test.

Figure 2.

Immediate shock does not regulate PSA-NCAM levels in the dentate gyrus, but context exposure upregulates its expression both in the dorsal and ventral parts. A, B, Number of polysialylated cells in the dentate gyrus 24 h after submitting animals either to a footshock (2 s; 1 mA) with minimal context exposure (A) or just to the context (B). Data represent mean ± SEM. **p < 0.01, ***p < 0.001 versus naive.

Figure 3.

Training increases the number of PSA-NCAM-labeled cells in the dentate gyrus 24 h after contextual fear conditioning. Left, Quantitative analysis of polysialylated cell number in the DG of two independent groups of animals, both of them trained in the contextual fear conditioning (1 mA), but one of them also tested 24 h later (Training + Test 24 h) and the other left undisturbed after training (Only training). Both groups of animals were perfused 24 h after training. Right, The same experimental protocol was followed to check independently the effects of training and retrieval in the tone fear-conditioning protocol on the number of hippocampal PSA-NCAM-labeled cells. Data represent mean ± SEM. Thickest horizontal discontinuous line represents mean values of the naive group, and thinner ones represent SEM values of this group. *p < 0.05 versus naive.

Figure 4.

Upregulation of PSA-NCAM-labeled cells in the hippocampus induced by acquisition of contextual fear conditioning is specifically circumscribed to the dorsal, but not ventral, subdivision of the dentate gyrus. A, Number of PSA-NCAM-immunoreactive cells in the granular cell layer of the dorsal dentate gyrus 24 h after training (1 mA) rats in a contextual (left) or in a tone (right) fear-conditioning paradigm. B, Number of PSA-NCAM-IR cells in the ventral subdivision of the dentate gyrus 24 h after contextual (right) or tone fear-conditioning (right) training (1 mA). C, Coronal brain sections illustrating the dorsal (left) and ventral (right) subdivisions of the hippocampal DG (red color) used to evaluate the frequency of cells expressing PSA-NCAM in these areas. Numbers indicate distance from bregma in millimeters. Data represent mean ± SEM. A, B, Thickest horizontal discontinuous line represents mean values of the naive group and thinner ones represent SEM values of this group. **p < 0.01 and ***p < 0.001 versus naive.

Figure 5.

Enzymatic removal of PSA-NCAM in the dorsal hippocampus disrupts long-term establishment of contextual fear conditioning. A, B, Bilateral endo-N injections given 24 h before training reduced freezing to the context in animals training under 0.4 mA (A) or 1 mA (B) shock intensity. C, D, Lack of effect of bilateral pretraining endo-N injections on freezing behavior during tone fear-conditioning tests. E, Distribution of PSA-NCAM immunoreactivity 4 d after endo-N injection. Shaded areas in the brain template show the representative diffusion of the enzyme across animals. Injection of endo-N effectively eliminated PSA-NCAM around the infusion site, covering the rostrocaudal extension of the hippocampus from the very rostral edge until 4–5 mm caudally. Diffusion of the enzyme throughout the hippocampus covered the whole dorsal dentate gyrus and CA1 regions and ∼73% of the dorsal CA3 region, without affecting the ventral edge. Numbers indicate distance from bregma in millimeters. F, G, Representative photomicrographs of a coronal section (top) and detail of the dentate gyrus (bottom) in an animal injected with endo-N or with vehicle, respectively. *p < 0.05 versus vehicle-injected group. Data represent mean ± SEM.

Figure 6.

Intact locomotion, emotional reactivity, and startle response after bilateral injection of endo-N in the dorsal hippocampus. A, Mean percentage ± SEM of time rats spent exploring center (A, left) and distance traveled (B, right) in the open field 48 h after enzyme injection. B, Mean amplitude ± SEM of startle response in two following sessions consisting of 10 randomly startle trials (115 dB; 40 ms) 72 h after endo-N administration.