Predictability and heritability of individual differences in fear learning

Abstract

Our objective was to characterize individual differences in fear conditioning and extinction in an outbred rat strain, to test behavioral predictors of these individual differences, and to assess their heritability. We fear-conditioned 100 Long-Evans rats, attempted to extinguish fear the next day, and tested extinction recall on the third day. The distribution of freezing scores after fear conditioning was skewed, with most rats showing substantial freezing; after fear extinction, the distribution was bimodal with most rats showing minimal freezing, but a substantial portion showing maximal freezing. Longer rearing episodes measured prior to conditioning predicted less freezing at the beginning of extinction, but differences in extinction learning were not predicted by any baseline exploratory behaviors. We tested the heritability of extinction differences by breeding rats from the top and bottom 20% of freezing scores during extinction recall. We then ran the offspring through the same conditioning/extinction procedure, with the addition of recording ultrasonic vocalizations throughout training and testing. Only a minority of rats emitted distress vocalizations during fear acquisition, but the incidence was less frequent in the offspring of good extinguishers than in poor extinguishers or randomly bred controls. The occurrence of distress vocalizations during acquisition predicted higher levels of freezing during fear recall regardless of breeding line, but the relationship between vocalization and freezing was no longer evident following extinction training, at which point freezing levels were influenced only by breeding and not by vocalization. The heritability (h(2)) of extinction recall was estimated at 0.36, consistent with human estimates.

Fig. 1

Probability density plots of the distributions of median freezing scores from the first 3 trials of the extinction session (a “Post-acquisition freezing”) and from the 3 probe trials conducted 24 h later (b “Post-extinction freezing”). Separate distributions are shown for 50 males obtained from Charles River and 50 females obtained from Harlan

Fig. 2

Extinction curves for rats classified as high (HE Phenotype) versus low (LE Phenotype) extinguishers based on differences at 24-h recall. Freezing to context was not significantly different between the two phenotypes, but there was a significant difference in freezing throughout extinction (repeated measures main effect, P < .001)

Fig. 3

Scatter plots of offspring conditioned freezing as a function of their parents’ conditioned freezing 24 h post-acquisition and 24 h post-extinction. Each point represents a single family with the parent mean plotted on the X axis and the offspring mean plotted on the Y axis

Fig. 4

Scatter plot showing the composition of each litter in terms of the percentage of offspring showing an LE phenotype (>75 % freezing before extinction and >75 % freezing after extinction) versus the percentage showing an HE phenotype (>75 % freezing before extinction and <25 % freezing after extinction). Note that the axes do not represent freezing scores. Rather, they reflect the percentage of offspring in each litter that actually met the specified criteria of being LE or HE, regardless of whether they were born to an LE or HE parent. Each point represents a litter. The shading indicates whether the parents were LE, HE, or RB (see legend). The X coordinate indicates what percentage of the litter actually displayed an LE phenotype, and the Y coordinate indicates what percentage of the litter actually displayed an HE phenotype. This graph illustrates the variability in the response to selection: in any given litter, some rats show the phenotype of their parents, some show the opposite phenotype, and the remainder are somewhere in between

Fig. 5

a Histogram showing frequency of 18–32 kHz “negative affect” vocalizations. b Mean 18–32 kHz vocalizations of the vocalizer sample (excluding non-vocalizers) as a function of training. Each trial (T1, T2, T3) is subdivided into three 20-s intervals before, during, and after the tone CS (Pre-CS, CS, and Post-CS). c Spectrogram from a single vocalizing subject for a single acquisition trial showing vocalizations at approximately 20 kHz, the 5 kHz tone CS, and the animal’s reaction to the shock US, which appears as a vertical line of broad-spectrum energy at the end of the tone

Fig. 6

Freezing as a function of heredity, vocalization, and training. Breeding line is indicated by shading (see legend), and vocalization group (silent vs. vocal in emitting ~22 kHz sounds during acquisition) is indicated by separate panels. Means are plotted as lines with 95 % confidence bands. Vocalizers showed significantly more freezing after acquisition, but not after extinction. Breeding lines were significantly different after extinction, but not after acquisition, p < .05