Auditory trace fear conditioning requires perirhinal cortex

Abstract

The hippocampus is well-known to be critical for trace fear conditioning, but nothing is known about the importance of perirhinal cortex (PR), which has reciprocal connections with hippocampus. PR damage severely impairs delay fear conditioning to ultrasonic vocalizations (USVs) and discontinuous tones (pips), but has no effect on delay conditioning to continuous tones. Here we demonstrate that trace auditory fear conditioning also critically depends on PR function. The trace interval between the CS offset and the US onset was 16s. Pre-training neurotoxic lesions were produced through multiple injections of N-methyl-D-aspartate along the full length of PR, which was directly visualized during the injections. Control animals received injections with phosphate-buffered saline. Three-dimensional reconstructions of the lesion volumes demonstrated that the neurotoxic damage was well-localized to PR and included most of its anterior-posterior extent. Automated video analysis quantified freezing behavior, which served as the conditional response. PR-damaged rats were profoundly impaired in trace conditioning to either of three different CSs (a USV, tone pips, and a continuous tone) as well as conditioning to the training context. Within both the lesion and control groups, the type of cue had no effect on the mean CR. The overall PR lesion effect size was 2.7 for cue conditioning and 3.9 for context conditioning. We suggest that the role of PR in trace fear conditioning may be distinct from some of its more perceptual functions. The results further define the essential circuitry underlying trace fear conditioning to auditory cues.

Figure 1. Training paradigm and spectrograms of the three conditional stimuli (CSs) used as cues

(A) A continuous 23 kHz tone. (B) A discontinuous 23 kHz tone (“pips”) whose frequency and on/off temporal pattern matches the ~23 kHz ultrasonic vocalization (USV). (C) A segment of a ~23 kHz USV recorded from a conspecific. All cues were matched in principle frequency, duration, and average loudness. (D) Training paradigm. Training included 10 trials of 9.7 s CS presentation, followed by a 16 s trace interval, and terminated with a 1 s US footshock presentation.

Figure 2. Examples of histology from a sham-operated subject (A–C) and a PR-lesioned subject (D – F)

Coronal slices were reacted using a Nissl stain (A, D), which labels neuronal and glial cell bodies; a gold-chloride stain (B, E), which labels myelin; a NeuN (C, F), which is specific for neurons. The illustrated sections were taken at approximately −5.2 A/P relative to bregma. Multiple injections of PBS resulted in unremarkable damage primarily represented by the needle tracks, while multiple injections of NMDA caused a well-localized neuronal damage. (G) Unilateral images showing the anterior-posterior extent of a representative PR lesion. Brain sections belong to the same subject shown in (D–F).

Figure 3. Average amount of bilateral damage to PR and adjacent structures caused by neurotoxic lesions

The amount of damage is given as a percentage of the total volume. PR sustained extensive bilateral damage, whereas damage to adjacent brain regions was limited. In the LA/BLA region there was significantly more damage to the right side than on the left side. Abbreviations: PR, perirhinal cortex; LA/BLA, lateral and basolateral nuclei of amygdala; EC, entorhinal cortex; vHC, ventral hippocampus; TE, temporal cortex. Bars represent the standard error (SE) of the mean.

Figure 4. Effects of the bilateral NMDA lesions on freezing to the cue and training context

(A) Mean freezing levels to the cue in PR-lesioned subjects (black bars) and sham-operated control animals (white bars). All three cues elicited robust freezing in the sham-operated control animals. By contrast, freezing was significantly reduced to all auditory cues in PR-lesioned subjects. (B) Mean freezing levels to the training context were comparable among the three groups of sham-operated subjects. By contrast, freezing was significantly reduced in the three groups of PR-lesioned subjects. Asterisks denote significant differences between the sham-operated and PR-lesioned subjects. Bars represent the standard error (SE) of the mean.