Intrinsic neuronal excitability is reversibly altered by a single experience in fear conditioning

Abstract

Learning is known to cause alterations in intrinsic cellular excitability but, to date, these changes have been seen only after multiple training trials. A powerful learning task that can be quickly acquired and extinguished with a single trial is fear conditioning. Rats were trained and extinguished on a hippocampus-dependent form of fear conditioning to determine whether learning-related changes in intrinsic excitability could be observed after a few training trials and a single extinction trial. Following fear training, hippocampal slices were made and intrinsic excitability was assayed via whole cell recordings from CA1 neurons. Alterations in intrinsic excitability, assayed by the postburst afterhyperpolarization and firing frequency accommodation, were observed after only three trials of contextual or trace-cued fear conditioning. Animals that had been trained in contextual and trace-cued fear were then extinguished. Context fear-conditioned animals extinguished in a single trial and the changes in intrinsic excitability were reversed. Trace-cue conditioned animals only partially extinguished in a single trial and reductions in excitability remained. Thus a single learning experience is sufficient to alter intrinsic excitability. This dramatically extends observations of learning-specific changes in intrinsic neuronal excitability previously observed in paradigms requiring many training trials, suggesting the excitability changes have a basic role in acquiring new information.

Fig. 1.

Learning trace and contextual fear increases intrinsic excitability. A: training paradigm for Trained cohort. In each training session, Tone animals were presented with one tone (small white box), Shock animals were presented with one shock (small black dash), and Paired animals received a tone and shock separated by a 30-s “trace” stimulus-free interval. Naïve animals were not handled or exposed to the training context. B: Paired and Shock groups froze more across the training sessions and also froze significantly more than Tone animals in the last training session, indicating robust contextual learning. All bar graphs show means ± SE. C: cells from Paired and Shock animals had increased intrinsic excitability compared with Tone or Naïve animals as measured by the peak and 1-s amplitude of the afterhyperpolarization (AHP). Example traces from Paired and Tone animals are shown (peak AHP: Paired −4.70 ± 0.36 mV, Shock −4.23 ± 0.38 mV, Tone −5.62 ± 0.21 mV, Naïve −6.07 ± 0.31 mV; slow AHP: Paired −2.05 ± 0.19 mV, Shock −2.06 ± 0.24 mV, Tone −2.64 ± 0.15 mV, Naïve −3.07 ± 0.2 mV). D: cells from Shock and Paired animals showed decreased accommodation by firing more action potentials (APs) during a 1-s current step sufficient to produce 5 APs in the first 100 ms, with no difference in the current step (P = 0.23). Example traces from Paired and Tone animals are shown (Paired 22.4 ± 2.1 APs, Shock 21.7 ± 1.8 APs, Tone 16.3 ± 0.9 APs, Naïve 12.8 ± 1.1 APs).

Fig. 2.

A single testing session confirms learning, but also extinguishes contextual freezing. A: training paradigm for the Tested cohort. Testing sessions consisted of 226 s in the original context (light gray), with no other stimuli, followed 15 min later by exposure to the tone in a novel context (dark gray). B: Paired and Shock groups froze more across the training sessions and also froze significantly more than Tone animals in the last training session, indicating robust contextual learning. C: all animals from the Tested cohort were tested for contextual and cue learning. Freezing during the testing session of the Tested cohort revealed robust contextual learning in Shock and Paired groups compared with the Tone group. Only Paired animals exhibited freezing behavior in response to the cue. D: training paradigm for the Extinguished cohort. E: Paired and Shock groups froze more across the training sessions, indicating robust contextual learning. F: during the first testing session, freezing was the same as that for the Tested cohort—i.e., Shock and Paired groups learned the context, whereas only Paired learned the cue. In the second testing session, Paired animals continued to freeze to the context, but showed extinction to the cue. Shock animals exhibited extinction of contextual learning after only a single testing session; this was the time point at which physiologic recordings were made in the Tested cohort. Although it appears that the Shock animals froze less to the cue in the second testing session, this was not significant [repeated-measures ANOVA, F_(8,2) = 1.305, P = 0.29]. By the third testing session, extinction was complete for both groups.

Fig. 3.

Behavioral extinction in single or multiple trials is sufficient to reverse the learning-related increases in excitability. A: cells from Paired animals did not show any effect of a single testing trial and their excitability changes remained (peak: Trained −4.70 ± 0.36 mV; Tested −4.68 ± 0.34 mV). In Shock animals, a single testing trial reversed the reduction of the AHP seen after learning but before testing (peak: Trained −4.23 ± 0.38 mV; Tested −6.06 ± 0.32 mV). The AHP from these cells, after a single testing trial, was no different from that from Naïve cells (peak: −6.06 ± 0.31 mV) or from that of animals from the Extinguished cohort who showed complete extinction (peak: −5.77 ± 0.37 mV). B: after a single testing trial, the learning-related reduction of firing frequency accommodation was also reversed in the Shock group, as measured by firing fewer APs during a 1-s current injection sufficient to produce 5 APs in the first 100 ms (Trained: 21.7 ± 1.8 APs; Tested: 15.6 ± 1.5 APs). After a single testing session, firing frequency accommodation in the Shock group was no different from that of Naïve animals (12.8 ± 1.1 APs) or from that of animals from the Extinguished cohort (15.9 ± 1.2 APs). The Paired group showed no effect of a single testing session on firing frequency accommodation (Trained: 22.4 ± 2.1 APs; Tested: 19.8 ± 2.0 APs).