Genetic Depletion of BDNF Impairs Extinction Learning of a Spatial Appetitive Task in the Presence or Absence of the Acquisition Context

Abstract

Brain derived neurotropic factor (BDNF) supports neuronal survival, growth, and differentiation and is involved in forms of hippocampus-dependent and independent learning, as well as hippocampus-dependent learning. Extinction learning comprises active inhibition of no-longer relevant learned information, in conjunction with a decreased response of a previously learned behavior. It is highly dependent on context, and evidence exists that it requires hippocampal activation. The participation of BDNF in memory processing is experience-dependent. For example, BDNF has been associated with synaptic plasticity needed for spatial learning, and it is involved in acquisition and extinction learning of fear conditioning. However, little is known about its role in spatial appetitive extinction learning. In this study, we evaluated to what extent BDNF contributes to spatial appetitive extinction learning in the presence (ABA) or absence (AAA) of exposure to the acquisition context. Daily training, of BDNF^+/--mice or their wildtype (WT) littermates, to reach acquisition criterion in a T-maze, resulted in a similar performance outcome. However, extinction learning was delayed in the AAA, and impaired in the ABA-paradigm compared to performance in WT littermates. Trial-by-trial learning analysis indicated differences in the integration of the context into extinction learning by BDNF^+/--mice compared to WT littermates. Taken together, these results support an important role for BDNF in processes that relate to information updating and retrieval that in turn are crucial for effective extinction learning.

Keywords: AAA paradigm; ABA paradigm; BDNF; appetitive; context-dependent; extinction learning; rodent; spatial learning.

FIGURE 1

Genotyping, Western blots and schema of the behavioral protocol. (A) Genotyping of BDNF mice. In BDNF^+/− animals, a neomycin-resistant gene, surrounded by a glycerate kinase gene promotor and a polyadenylation signal, replaces one allele of the BDNF protein-coding exon. Most of the mature BDNF is therefore depleted, and the introduced gene serves as a biomarker. BDNF^+/− animals (bands 3 and 4, from the left in each gel) expressed both the neomycin-resistant gene (500 bp) as well as BDNF (500 bp), whereas in the wild type (WT, second band from the left) animals only the BDNF bands are observable. – and ++ bands correspond to negative and positive controls, respectively, for both genes. (B) Hippocampal BDNF expression is depleted in the BDNF^+/− animals. The expression of BDNF (14 kDa) and β-actin (42 kDa) protein in the hippocampus of both BDNF^+/− (n = 4) and wild type littermates (WT) animals (n = 4) was evaluated by Western blot. β-Actin was used as a protein loading control, and each individual value was normalized by the β-Actin expression level. Expression of mature BDNF was significantly lower in the transgenic animals as compared to their WT littermates. Group averages (±S.E.M) are indicated by vertical bars, points represent individual measurements (p = 0.006). (C) Schema of the behavioral protocol. Top: On Day 1, animals participate in two sessions each comprising ten consecutive trials each (separated by 10-min intervals) that include a reward probability of 100%. On Day 2 two ten trial sessions at 80% of reward probability occur. On Day 3, the reward probability of the first session is 80%, and of the second session is 60%. On Day 4, the reward probability declines from 60% in the first session, to 30% in the second session. Bottom right: On Days 5 and 6, animals participate in the extinction learning protocol in the same context (AAA) in the absence of a reward. Bottom left: In the ABA paradigm animals engage in extinction learning in the presence of a context change on Day 5. During extinction learning, the context (floor pattern, distal cues, odor cues) are changed. On Day 6, renewal is tested by re-exposure to context A. No reward is present in the maze during the extinction or renewal test days.

FIGURE 2

BDNF^+/− mice do not exhibit impairments in task acquisition. The bar charts show the percentage of correct choices made by the animals during task acquisition learning over 4 days in context A. Each bar pair shows the animals’ correct choice performance in a given session (S) on a specific day (D) of acquisition. White bars show the response of wild type (WT) mice and gray bars show the response of transgenic BDNF^+/− mice. The dots (black: WT, gray outlined: BDNF^+/−) show the distribution of choice performance in a specific session. Both WT (n = 16) and BDNF^+/− animals (n = 16) acquired the task over the 4 days, as signified by the increasing percentage of correct responses in both groups. Despite the gradual reduction in the reward probability, the two groups reached the 85% criterion of correct responses (dashed line) by day 4.

FIGURE 3

BDNF^+/− mice exhibit a delay in extinction learning in the AAA context and an impairment in extinction learning in the ABA context. The bar charts show the percentage of correct choices made by the animals during task acquisition on Day 4 involving sessions 1 and 2 in context A. In addition, arm choice performance is shown during the 2 days of extinction trials in the same context as used for acquisition (AAA paradigm) involving two training sessions per day (D5S1-D6S2). Furthermore, arm choice performance is shown when extinction trials occur in a novel context (compared to the acquisition conditions) during two sessions on Day 5 (D5S1, D5S2), and when animals are re-exposed to the original acquisition context during two sessions on Day 6 (D6S1, D6S2) (ABA paradigm). Each bar pair shows the animals’ correct choice performance. The dots (black: WT, gray outlined: BDNF^+/) show the distribution of choice performance in a specific session, and the dashed line represents the chance performance level. (A) AAA paradigm: Although correct choices in BDNF^+/− mice (n = 8) and their WT littermates (n = 8) were equivalent during both acquisition learning sessions on Day 4 (D4S1, D4S2) when extinction learning was assessed on Day 5, a significant difference between extinction learning performance in WT and BDNF^+/− mice became apparent. WT mice showed rapid extinction learning (D5S2, D6S1), whereas effects in BDNF^+/− mice were significantly slower. By end of Day 6, BDNF^+/− mice exhibited extinction learning that was not significantly different from WT littermates, performing at chance levels (dashed line). (B) In the ABA paradigm, BDNF^+/− mice (n = 8) and their WT littermates (n = 8) also displayed equivalent levels of correct choices during both acquisition learning sessions in context A on Day 4 (D4S1, D4S2). However, when extinction learning was tested in context B on Day 5, a significant impairment in extinction learning became evident in BDNF^+/−mice compared to WT littermates (whereas WT mice exhibit performance differences between the end of acquisition, D4S2 to D5S1 and D5S2, BDNF^+/− do not). Although WT mice exhibited significant renewal on Day 6 (correct choices during D6S1 compared to choices during D5S2), BDNF^+/− mice displayed choice levels during D6S1, that were not different from choices made on Day 5, indicating that WT exhibited renewal. By contrast, BDNF^+/− mice showed perseverance in entering the previously rewarded target arm. *(p ≤ 0.05) and ⁺(p ≤ 0.01).

FIGURE 4

Comparison of trial-by-trial learning on days 5 and 6. To assess trial-by-trial learning, the number of correct arm choices made by each animal was plotted for each individual trial. (A) The number of animals entering the correct arm in context “A” on Day 5, consistently declined over the 20 trial period with regard to wild-type animals. By contrast, BDNF^+/− mice numbers remained consistently high. (B) The number of animals entering the correct arm in context “A” on Day 6 is equivalent in wild-type and BDNF^+/− mice. (C) When extinction learning is tested in context “B”, wild-type animals exhibit a rapid diminishment of choice behavior for the previously rewarded arm, whereas BDNF^+/− mice persist in entering the arm. (D) On day 6, animals were returned to the “A” context (in the absence of a reward). Here, WT animals show an increase of “correct” arm choices in the initial trials compatible with a renewal effect. This response then diminished corresponding to extinction learning as the animal understands that no reward will be made available. BDNF^+/− mice show arm choice behavior that is similar to Day 5, but toward the end of the trial block evidence of extinction learning begins to emerge.