Generalized vs. stimulus-specific learned fear differentially modifies stimulus encoding in primary sensory cortex of awake rats

Abstract

Experience shapes both central olfactory system function and odor perception. In piriform cortex, odor experience appears critical for synthetic processing of odor mixtures, which contributes to perceptual learning and perceptual acuity, as well as contributing to memory for events and/or rewards associated with odors. Here, we examined the effect of odor fear conditioning on piriform cortical single-unit responses to the learned aversive odor, as well as its effects on similar (overlapping mixtures) in freely moving rats. We found that odor-evoked fear responses were training paradigm dependent. Simple association of a condition stimulus positive (CS+) odor with foot shock (unconditioned stimulus) led to generalized fear (cue-evoked freezing) to similar odors. However, after differential conditioning, which included trials where a CS- odor (a mixture overlapping with the CS+) was not paired with shock, freezing responses were CS+ odor specific and less generalized. Pseudoconditioning led to no odor-evoked freezing. These differential levels of stimulus control over freezing were associated with different training-induced changes in single-unit odor responses in anterior piriform cortex (aPCX). Both simple and differential conditioning induced a significant decrease in aPCX single-unit spontaneous activity compared with pretraining levels while pseudoconditioning did not. Simple conditioning enhanced mean receptive field size (breadth of tuning) of the aPCX units, while differential conditioning reduced mean receptive field size. These results suggest that generalized fear is associated with an impairment of olfactory cortical discrimination. Furthermore, changes in sensory processing are dependent on the nature of training and can predict the stimulus-controlled behavioral outcome of the training.

Fig. 1.

Olfactory fear conditioning with different paradigms. A: odor mixtures used in the experiments. 10C, an odor mixture that consists of 10 different odorants; 10C-1, 10C with 1 odorant missing; 10C-2, 10C with 2 odorants missing; 10CR1, 10C with 1 odorant replaced by a new odorant; NC, no change. B: odor-fear training paradigms. Standard training had 10 trials of 10C paired with an electric foot shock. Pseudotraining had 10 unpaired presentations of 10C and foot shock. Differential training had 10 trials of 10C [conditioned stimulus positive (CS+)] paired with a foot shock and 10C-2 (CS−) without a following foot shock. A retention test was carried out 24 h after the training session. All rats were randomly presented with 10C, 10C-1, 10C-2, 10CR1, and limonene 3 times. Odor-evoked freezing behavior of the rats was scored and recorded. C: average freezing of the nonimplanted rats in response to odor stimuli 24 h after training. Standard, differential, and pseudo represent different training paradigms. Bars that are marked with “a” are significantly different from bars that are marked with “b” and “c” [P < 0.05, Fisher's paired least significant difference (PLSD)]. D: results from a second behavioral test following 3 days of reminder training (3 group appropriate odor-shock pairings/day) as was also performed in the animals used for electrophysiological recording. No change in the overall pattern of results in the 3 groups was observed. Error bars represent SE.

Fig. 3.

Anterior piriform cortex (aPCX) single-unit recordings from awake, freely moving rats. A: a rat implanted with a movable wire bundle was in the testing chamber for chronic unit recording and odor training. Gray bars in the chamber represent the metal grid floor that was part of the electric shocking system. B: procedures of chronic unit recordings. Note that each rat only was only trained once on a single day, while recording sessions were conducted for multiple days before and after the training day. C: a digital-filtered (bandpass, 300–3,000 Hz) trace showing spikes from a single microwire placed in aPCX. One larger and one smaller spike can be seen. Trace has a time base of 5 ms per division. D: extracted waveforms of units 1 and 2 from the trace in C. Signal-to-noise ratio of unit 1 (left) was 2.5:1; that of unit 2 (right) was 6:1. E: examples of peristimulus time histograms with raster plots of 2 single units (left and right) in response to 10CR1. Histograms showed cumulative spike count of three trials, with a 100-ms bin width. Both units showed a significant excitatory response to 10CR1. Horizontal black bars indicate odor delivery (5 s).

Fig. 2.

Representation of electrode tracks of 15 rats implanted with a movable microwire bundle. Black bars represent electrode tracks reconstructed from the brain sections. Recording sites were along the tracks. Black open rectangle represents possible recording sites across layer III of piriform cortex where track reconstruction was not available. Data suggested recordings were localized to layer II/III of anterior piriform cortex. Outlines are reproduced from Paxinos and Watson (2009) Copyright Elsevier, and represent sections ranging from 2.70 to 0.48 mm anterior to Bregma.

Fig. 4.

Effects of odor-fear conditioning on spontaneous activity of aPCX neurons. Standard and differential training induced a significant decrease in spontaneous firing rate of neurons in the aPCX. This decrease was not significant in the pseudotrained group. There was no significant difference in spontaneous activity of the animals before training. Error bars represent SE. *P < 0.01.

Fig. 5.

Proportion of single units responsive to odors before and after training in the three groups. A: proportion of single units showing response to odor (excitation + suppression) was enhanced after standard conditioning and decreased after differential conditioning, while pseudoconditioning induced no change. B: standard and differential training induced proportionally less excitatory single-unit odor responses to the test odors, while pseudoconditioning induced no change. C: standard training induced proportionally more suppressive odor responses, with no change after either pseudo or differential conditioning. The change in proportion of units responding to odors [total (D), excitatory (E), and suppressive (F)] after standard conditioning was not odor specific. Error bars represent SE. *P < 0.05. For calculation of single-unit odor responses, see materials and methods.

Fig. 6.

Single-unit receptive field (RF) width before and after training in the three groups. A: standard training induced a significant increase in mean RF size of anterior piriform cortex neurons. In contrast, differential training induced a significant decrease in mean RF size. No change was induced by the pseudotraining. B: standard and differential training induced a significant decrease in excitatory RF size. No change was observed after pseudotraining. C: standard training induced a significant increase in suppressive RF size, while no change in suppressive RFs was observed after pseudo or differential conditioning. Error bars represent SE. *P < 0.05. For receptive field calculation, see materials and methods.