The experimental paradigm used in the present study is shown in . Lewis rats received vehicle (n = 28) or ibotenic acid infusions (n = 40) aimed to the BNST and were allowed to recover for one week. BNST-lesioned and sham rats then underwent the same protocol in parallel. After habituation (, Day-0), they were subjected to a differential fear conditioning paradigm (, Day-1) where one of two tones (CS+) was paired to a footshock whereas a second tone (CS−) was not. On Day-2, freezing to the context was assessed. Recall of cued fear was tested on Day-3 in a different context. Two days after the recall test, anxiety levels were assessed using an elevated plus maze (EPM). At the conclusion of the experiments, the animals were perfused-fixed, their brains sectioned, and the sections processed to reveal NeuN immunoreactivity () to assess the location and specificity of the lesions. On this basis, we restricted our attention to the behavior of a subset of 18 animals that had extensive and selective BNST lesions. See supplementary figure 1
for the extent of the largest and smallest BNST lesions included in this study.
and illustrate in parallel the results obtained in sham (black lines and symbols) and BNST-lesioned (red lines and symbols) rats. Filled and empty circles represent freezing to the CS+ and CS−, respectively. Diamonds represent contextual freezing in the absence of CS. To facilitate explanation of the data, we first describe the results obtained in sham rats and then contrast their behavior to that of BNST-lesioned animals.
Fig. 2 BNST lesions abolish behavioral heterogeneity related to discrimination abilities. (A) Percent time freezing in response to the CS+ (filled circles) and CS− (empty circles) in individual sham (black; n = 28) vs. BNST-lesioned (red; n = 18) rats (more ...)
Sham rats exhibited normal acquisition of conditioned fear responses to the CS+ (Day 1), significant freezing to the training context (Day 2), and excellent fear recall (Day 3; ). Although they exhibited poor discrimination between the CS+ and CS− during conditioning and recall, freezing to the CS− was significantly lower than to the CS+ during recall (t-test, first CS+ vs. CS−, p=0.001). However, there was considerable inter-individual heterogeneity in fear responding among sham rats, particularly during recall (, black). Although all sham rats exhibited high freezing in response to the CS+, freezing to the CS−was widely distributed, with some animals freezing as much to the CS− as to the CS+, and others freezing much less to the CS−. Thus, although the collective behavior of sham rats indicates poor discrimination between the CS+ and CS−, inter-individual variations suggest a more complex situation where some subjects exhibit good discrimination between the two CSs whereas others generalize fear to the safe tone (CS−).
Next, we determined whether the varying aptitude to discriminate between the two CSs seen in sham rats was associated with other behavioral properties. To this end, we computed a frequency distribution of the difference in freezing to the first CS+ and CS− of the recall test, hereafter termed discrimination score, operationally defined as a measure of discriminative ability. The ability to discriminate between the two CSs was not distributed normally among sham rats, the frequency distribution being highly skewed to the right (, black). On the basis of this distribution, we used a cut-off of 30% difference in discrimination score to distinguish sham rats with low vs. high discriminative abilities.
No difference in the amount of freezing to the CS+ was found between the two subsets of sham rats during recall (; p>0.05; t-test comparing freezing to the first CS+). In contrast, sham rats with a high discrimination score exhibited significantly less freezing to the training context (, Ctx; 24.0±4.4%) than poor discriminators (45.1±5.4%, p=0.03). In keeping with this, discrimination score and context freezing were inversely correlated among sham rats (; r=−0.49, p<0.05). Moreover, poor discriminators spent significantly more time in the closed arms of the EPM than sham rats with good discriminative abilities (, EPM; t-test, p=0.03), these two variables being inversely correlated (; r=−0.54, p<0.05).
Thus, the above analysis suggests that sham rats exhibit a continuum of anxious/fearful behaviors where different measures of fear and anxiety tend to co-vary. At one pole of the distribution are rats that display poor discriminative abilities, high contextual freezing, and a marked anxiety-like trait in the EPM. At the other pole are rats that display less fear generalization to the CS−, lower freezing to context, and a non-anxious trait on the EPM. See supplementary Fig. 2
Insights into BNST contributions to these inter-individual variations can be gained by comparing the behavior of sham vs. BNST-lesioned rats. Since the ability to discriminate between the CS+ and CS− during recall was most predictive of fearful/anxious behavior in sham rats, we first compare the two groups along this dimension. Discrimination ability during the recall test was drastically higher in rats with BNST lesions (, , red) than in sham rats (, , black). Indeed, 17 of 18 BNST-lesioned animals had a DS > 30% (). The differing proportion of rats with low or high DSs in sham vs. BNST-lesioned rats was significant (Fisher Exact test, p=0.00006). So were the average DSs of sham (21.7±4.9%) and BNST-lesioned rats (53.7±3.9%; t-test, p=0.00004). This difference was due to decreased freezing to the CS− since behavior during the CS+ did not differ between the two groups (, t-test, p=0.52).
Since discriminative ability was predictive of anxious/fearful behavior in the sham animals, we next examined whether BNST-lesioned animals behaved like sham rats with high DSs (). In keeping with this, BNST-lesioned rats were statistically indistinguishable from sham rats with high discriminative abilities in the amount of contextual freezing they displayed (; t-test, p=0.14) and time spent in the closed arms of the EPM (t-test, p=0.45). The marked reduction in contextual freezing seen in rats with BNST lesions is consistent with previous reports (Sullivan et al., 2004
; Resstel et al., 2008