Interfering With Contextual Fear Memories by Post-reactivation Administration of Propranolol in Mice: A Series of Null Findings

Abstract

Post-reactivation amnesia of contextual fear memories by blockade of noradrenergic signaling has been shown to have limited replicability in rodents. This is usually attributed to several boundary conditions that gate the destabilization of memory during its retrieval. How these boundary conditions can be overcome, and what neural mechanisms underlie post-reactivation changes in contextual fear memories remain largely unknown. Here, we report a series of experiments in a contextual fear-conditioning paradigm in mice, that were aimed at solving these issues. We first attempted to obtain a training paradigm that would consistently result in contextual fear memory that could be destabilized upon reactivation, enabling post-retrieval amnesia by the administration of propranolol. Unexpectedly, our attempts were unsuccessful to this end. Specifically, over a series of experiments in which we varied different parameters of the fear acquisition procedure, at best small and inconsistent effects were observed. Additionally, we found that propranolol did not alter retrieval-induced neural activity, as measured by the number of c-Fos⁺ cells in the hippocampal dentate gyrus. To determine whether propranolol was perhaps ineffective in interfering with reactivated contextual fear memories, we also included anisomycin (i.e., a potent and well-known amnesic drug) in several experiments, and measures of synaptic glutamate receptor subunit GluA2 (i.e., a marker of memory destabilization). No post-retrieval amnesia by anisomycin and no altered GluA2 expression by reactivation was observed, suggesting that the memories did not undergo destabilization. The null findings are surprising, given that the training paradigms we implemented were previously shown to result in memories that could be modified upon reactivation. Together, our observations illustrate the elusive nature of reactivation-dependent changes in non-human fear memory.

Keywords: contextual fear; hippocampus; memory reconsolidation; propranolol; rodents.

Figure 1

Noeffect of propranolol on reactivated memories that were acquired witha single 0.7 mA shock during contextual fear conditioning.(A) Design of Experiment 1. Mice received one 0.7 mA shockduring fear conditioning. One day later, saline (n = 8) orpropranolol (n = 12) was administered after memoryreactivation. Forty-eight hours afterwards, retention of contextualfear was assessed. Cond, conditioning; MR, memory reactivation;Ret, retention; CS, conditioned stimulus (context); Sal, saline;Prop, propranolol. (B) Results of Experiment 1a. Averagepercentage of freezing during memory reactivation is displayed on theleft, and freezing during the retention test on the right panel ofthe column chart (saline in white bars, propranolol in red bars).Error bars represent SEM. Filled circles indicate individual animals.(C) Results of Experiment 1b (saline, n = 9;propranolol, n = 10). Experiment 1b was a direct replication of Experiment 1a. (D) Collapsed results of Experiment 1a and Experiment 1b. (E) Representative images of the DG 90 min after the retention test from mice that received either saline (n = 5), or propranolol (n = 5) after reactivation (Experiment 1a). Cells that were c-Fos⁺ are labeled in cyan (scale bar: 200 μm). (F) Number of c-Fos⁺ cells in the DG per 1.3 mm². Error bars represent SEM. Filled circles indicate individual animals.

Figure 2

Aminor effect of propranolol on reactivated memories that were acquired with a single 1 mA shock during contextual fear conditioning. (A) Design of Experiment 2a. Mice received one 1 mA shock during fear conditioning. One day later, saline (n = 8) or propranolol (n = 8) was administered after memory reactivation. Forty-eight hours afterwards, retention of contextual fear was assessed. Cond, conditioning; MR, memory reactivation; Ret, retention; CS, conditioned stimulus (context);Sal, saline; Prop, propranolol. (B) Results of Experiment 2a. Average percentage of freezing during memory reactivation is displayed on the left, and freezing during the retention test on the right panel of the column chart (saline in white bars, propranolol in red bars). Error bars represent SEM. Filled circles indicate individual animals. (C) Results of Experiment 2b (saline, n = 9; propranolol, n = 9). Experiment 2b was a direct replication of Experiment 2a. (D) Collapsed results of Experiment 2a and Experiment 2b. (E) Representative images of the DG 90 min after the retention test from mice that received either saline (n = 7), or propranolol (n = 7) post-reactivation (Experiment 2a). Cells that were c-Fos⁺ are labeled in cyan (scale bar: 200 μm). (F) Number of c-Fos⁺ cells in the DG per 1.3 mm². Error bars represent SEM. Filled circles indicate individual animals.

Figure 3

Noeffect of propranolol—injected in two differentvolumes—on reactivated memories that were acquired with asingle 1 mA shock during contextual fear conditioning. (A)Design of Experiment 3. Mice received one 1 mA shock during fear conditioning. One day later, mice received saline (n = 9), propranolol (n = 9), or propranolol dissolved in saline at half the ratio and injected in double the volume/body weight (n = 9) after memory reactivation. Forty-eight hours afterward, retention of contextual fear was assessed. Cond, conditioning; MR, memory reactivation; Ret, retention; CS, conditioned stimulus (context); Sal, saline; Prop, propranolol. (B) Results of Experiment 3. Average percentage of freezing during memory reactivation is displayed on the left, and freezing during the retention test on the right panel of the column chart (saline in white bars, propranolol in red bars, and propranolol double volume in orange bars). Error bars represent SEM. Filled circles indicate individual animals.

Figure 4

Noeffect of propranolol on reactivated memories that were acquired witha single 1 mA shock during contextual fear conditioning in theanimals’ dark phase. (A) Design of Experiment 4. Micereceived one 1 mA shock during fear conditioning. One day later, mice received saline (n = 12) or propranolol (n = 12) after memory reactivation. Forty-eight hours afterward, retention of contextual fear was assessed. All procedures took place in the animals’ dark phase. Cond, conditioning; MR, memory reactivation; Ret, retention; CS, conditioned stimulus (context); Sal, saline; Prop, propranolol. (B) Results of Experiment 4. Average percentage of freezing during memory reactivation is displayed on the left, and freezing during the retention test on the right panel of the column chart (saline in white bars, propranolol in red bars). Error bars represent SEM. Filled circles indicate individual animals.

Figure 5

Noeffect of propranolol on reactivated memories in a backgroundcontextual fear-conditioning paradigm. (A) Design ofExperiment 5. Mice received one 1 mA shock during presentation of atone. One day later, mice received saline (n = 11) or propranolol (n = 11) after contextual memory reactivation. Forty-eight hours afterward, retention of contextual fear was assessed by exposure to the context. Cond, conditioning; MR, memory reactivation; Ret, retention; CS, conditioned stimulus (context); Sal, saline; Prop, propranolol. (B) Results of Experiment 5. Average percentage of freezing during memory reactivation is displayed on the left panel of the column chart, and freezing during the retention test on the right panel of the column chart (saline in white bars, propranolol in red bars). Error bars represent SEM. Filled circles indicate individual animals.

Figure 6

Noeffect of propranolol or anisomycin on reactivated memories that were acquired with a single 1 mA shock during contextual fear conditioning. (A) Design of Experiment 6. Mice received one 1 mA shock during fear conditioning. One day later, mice received saline (n = 8), propranolol (n = 9), or anisomycin (n = 8) after memory reactivation. Forty-eight hours afterward, retention of contextual fear was assessed. Cond, conditioning; MR, memory reactivation; Ret, retention; CS, conditioned stimulus (context); Sal, saline; Prop, propranolol; Ani, anisomycin. (B) Results of Experiment 6. Average percentage of freezing during memory reactivation is displayed on the left, and freezing during the retention test on the right panel of the column chart (saline in white bars, propranolol in red bars, and anisomycin in blue bars). Error bars represent SEM. Filled circles indicate individual animals.

Figure 7

No effect of reactivation on synaptic GluA2 expression. (A) Design of Experiment 7. Mice received either no shock, one 0.7 mA shock, or one 1 mA shock during fear conditioning. One day later, mice underwent memory reactivation and received either saline (no shock, n = 5; 0.7 mA, n = 6; 1 mA, n = 6) or propranolol (no shock, n = 5; 0.7 mA, n = 5; 1 mA, n = 5) afterwards. One hour later the mice were sacrificed. Cond, conditioning; MR, memory reactivation; CS, conditioned stimulus (context); Sal, saline; Prop, propranolol. (B) Freezing levels during reactivation for the no shock group (left), 0.7 mA group (middle, dark gray), and 1 mA (right, black) group. (C) Results of Experiment 7. GluA2 expression levels relative to the no shock + saline control group (saline in white bars, propranolol in red bars). Error bars represent SEM. Filled circles indicate individual animals. (D) Upper panel: representative immunoblots for GluA2 with approximate molecular weight indicated. Lower panel: corresponding gels of total protein that were used for normalization.

Figure 8

No effect of propranolol or anisomycin on reactivated memories that were acquired with three 0.5 mA shocks during contextual fear conditioning. (A) Design of Experiment 8. Mice received three 0.5 mA shocks during fear conditioning. One day later, mice received saline (n = 9), propranolol (n = 9), or anisomycin (n = 9) after memory reactivation. Forty-eight hours afterward, retention of contextual fear was assessed. Cond, conditioning; MR, memory reactivation; Ret, retention; CS, conditioned stimulus (context); Sal, saline; Prop, propranolol; Ani, anisomycin. (B) Results of Experiment 8. Average percentage of freezing during memory reactivation is displayed on the left, and freezing during the retention test on the right panel of the column chart (saline in white bars, propranolol in red bars, and anisomycin in blue bars). Error bars represent SEM. Filled circles indicate individual animals.

Figure 9

No effect of propranolol or anisomycin on reactivated memories that were acquired with three 0.3 mA shocks during contextual fear conditioning. (A) Design of Experiment 9a. Mice received three 0.3 mA shocks during fear conditioning. One day later, mice received saline (n = 9), propranolol (n = 9), or anisomycin (n = 9) after memory reactivation. Forty-eight hours afterward, retention of contextual fear was assessed. Cond, conditioning; MR, memory reactivation; Ret, retention; CS, conditioned stimulus (context); Sal, saline; Prop, propranolol; Ani, anisomycin. (B) Results of Experiment 9a. Average percentage of freezing during memory reactivation is displayed on the left, and freezing during the retention test on the right panel of the column chart (saline in white bars, propranolol in red bars, and anisomycin in blue bars). (C) Design of Experiment 9b. The anisomycin condition was replaced with propranolol dissolved in saline at half the usual ratio and injected in double the volume/body weight (n = 9 in all conditions). All other procedures were the same as Experiment 9a. (D) Results of Experiment 9b (saline in white bars, propranolol in red bars, and propranolol double volume in orange bars). (E) Collapsed results of Experiment 9a and 9b (propranolol in all volumes). Error bars represent SEM. Filled circles indicate individual animals.