Dominance status predicts social fear transmission in laboratory rats

Abstract

Acquiring information about stimuli that predict danger, through either direct experience or inference from a social context, is crucial for individuals' ability to generate appropriate behaviors in response to threats. Utilizing a modified demonstrator-observer paradigm (fear conditioning by proxy) that allows for free interaction between subjects, we show that social dominance hierarchy, and the interactive social behaviors of caged rats, is predictive of social fear transmission, with subordinate rats displaying increased fear responses after interacting with a fear-conditioned dominant rat during fear retrieval. Fear conditioning by proxy conserves some of the pathways necessary for direct fear learning (e.g., lateral amygdala) but is unique in that it requires regions necessary for emotional regulation (e.g., anterior cingulate cortex), making this paradigm an important tool for evaluating learning and behavior in the laboratory setting.

Keywords: Dominance; Fear conditioning; Play behavior; Social learning.

Fig. 1

Experiments 1 and 2 design. a Experiment 1: Play behavior within a triad was observed 3 weeks prior to fear conditioning by proxy paradigm and used to determine dominance status. One rat of the triad was fear conditioned directly on day 1 (FC). On day 2, the FC rat and a cage-mate (FCbP rat) were exposed to the CS together. On day 3, each rat was exposed to the CS alone as a measure of long-term fear memory. Trunk blood was collected 30 min after LTM test on day 3 for analysis of serum corticosterone levels. b Experiment 2 design and counting frames for c-Fos IHC (circles). Rectangle indicates imaging frame from camera. One rat in the triad was fear conditioned to a tone CS on day 1 (FC Rat). The following day, the FC rat and FCbP rat were placed in the chamber together, the third No FC rat was placed in the chamber alone, while the CS was presented and rats were euthanized 1 h after cue presentation. Immunopositive nuclei were counted in the anterior cingulate cortex (ACC), the lateral nucleus of the amygdala (LA), the CA1 region of the dorsal hippocampus (dCA1) and the ventral portions of the CA2, CA3, and CA1 regions of the hippocampus. Circles represent the fixed counting frames used for cell quantification, counts were sampled from circles outlined in red (color figure online)

Fig. 2

Experiments 3 and 4 design. a Experiment 3: Experimental design for muscimol infusions into vHPC or ACC. Rats designated as FCbP (S2 rats) were surgically implanted with bilateral cannula aimed at either the ventral hippocampus or the anterior cingulate cortex and allowed to recover. Twenty minutes prior to fear conditioning by proxy with the FC rat, FCbP rats were infused with either saline or muscimol. The following day, in the absence of the drug, all rats were tested for freezing to the cues. b Experiment 4: experimental design. Rats were surgically implanted with bilateral guide cannulae aimed at either the vHPC or ACC and allowed to recover. Muscimol or saline was infused prior to direct CS–US pairings, and rats were tested for freezing to the CS the following day

Fig. 3

Subordinate FCbP rats froze during CS presentation on day 3 when paired with higher ranked fear expressing cage-mates. a–c Freezing on day 3 in each dominance subtype. There was no effect of dominance status on freezing after direct FC (far left bars of each panel). a The D FCbP rats did not freeze significantly more to the cues than D No FC rats after fear conditioning by proxy with either S1 (blue bars, n = 12) or S2 (white bars, n = 11) (both Ps > .05). b S1 FCbP rats froze significantly more after fear conditioning by proxy with either a FC D rat (black bar; n = 10) or S2 rat (white bar; n = 10) than S1 rats that were not fear conditioned (Ps < .05) but C) S2 rats only showed socially acquired freezing after fear conditioning by proxy with a FC D rat (black bar; n = 9) (P < .05). Error bars ± SEM (color figure online)

Fig. 4

Social behaviors as predictors of socially acquired fear. a–c Relationships between social behaviors and day 3 freezing in FCbP rat. a Nape contacts initiated by the FCbP rat toward the FC rat as a percentage of total nape contacts and b likelihood of evasion when FCbP nape contacts FC rat (percent of nape contacts that resulted in evasive response of FC rat) during the play behavior session were entered in the first step and contributed a small but significant amount. c Social contact during the cues of the fear conditioning by proxy session accounted for the largest amount of unique variance in LTM freezing displayed by the FCbP rat the following day

Fig. 5

Infrequent negative-affect ultrasonic vocalizations correlate with socially transmitted fear. a Sample spectrogram of a 22 kHz vocalization. b–d Frequency histograms of the number of subjects that elicited negative-affect vocalizations in the 22 kHz range, represented graphically on a logarithmic scale, during b fear conditioning on day 1, c fear conditioning by proxy on day 2, and d long-term memory tests on day 3 indicate that most rats do not vocalize in the 22 kHz range at all and only directly fear-conditioned rats vocalize on day 3. e Of the rats that do vocalize (n = 10), the total duration of 22 kHz calls during the FCbP session was positively correlated with freezing displayed by the FCbP rat the following day (P < .05)

Fig. 6

Serum corticosterone on Day 3 correlates with freezing on day 3 and dominance status. a FC rats (n = 39) had significantly higher corticosterone values than No FC rats (n = 37). When these values were further divided based on dominance assignment (inset), No FC S2 rats (n = 12) had significantly increased corticosterone than No FC D rats (n = 12). b Freezing on day 3 was moderately but significantly correlated with serum corticosterone levels measured 30 min after LTM test (n = 116)

Fig. 7

Increased c-Fos activity in ACC and vCA3 on day 2 in FCbP rats. a The ACC and vCA3 were uniquely activated in FCbP acquisition. Retrieval of a direct FC memory and acquisition of FCbP both activated the LA and vCA1. Error bars ± SEM. b Social contact on day 2 predicted c-Fos activity in the ACC in FCbP rats (n = 10) but not FC rats (n = 11)

Fig. 8

Temporary inactivation of ACC but not vHPC prior to FCbP prevents social fear transmission. a Cannula placement in the ventral hippocampus, b temporary inactivation of the vHPC did not influence freezing on day 3 after FCbP (saline n = 4; muscimol n = 5). c Cannula placement in ACC. d Freezing on day 3 LTM tests indicates that inactivation of the ACC prevented fear acquisition through the FCbP paradigm (saline n = 10; muscimol n = 8)

Fig. 9

Temporary inactivation of vHPC but not ACC prior to direct FC reduces freezing during LTM tests. a Locations of cannula tips in ventral hippocampus. b Freezing during long-term memory tests 24 h after infusion of either muscimol (n = 5) or saline (n = 5). Rats infused with muscimol into the vHPC froze less than saline rats to both the cues and in the 20 s immediately preceding the first CS presentation. c Location of cannula tips in the ACC. d Freezing to cues and 20 s pre CS presentation 24 h after direct fear conditioning and infusion of saline (n = 5) or muscimol (n = 7) indicate no differences in direct fear retention