Pheromone-Induced Odor Associative Fear Learning in Rats

Abstract

Alarm pheromones alert conspecifics to the presence of danger. Can pheromone communication aid in learning specific cues? Such facilitation has an evident evolutionary advantage. We use two associative learning paradigms to test this hypothesis. The first is stressed cage mate-induced conditioning. One pair-housed adult rat received 4 pairings of terpinene + shock over 30 min. Ten minutes after return to the home cage, its companion rat was removed and exposed to terpinene. Single-housed controls were exposed to either terpinene or shock only. Companion rats showed terpinene-specific freezing, which was prevented by β-adrenoceptor blockade. Using Arc to index neuronal activation in response to terpinene re-exposure, stressed cage-mate induced associative learning was measured. Companion rats showed increased neuronal activity in the accessory olfactory bulb, while terpinene + shock-conditioned rats showed increased activity in the main olfactory bulb. Both groups had enhanced activity in the anterior basolateral amygdala and central amygdala. To test involvement of pheromone mediation, in the 2nd paradigm, we paired terpinene with soiled bedding from odor + shock rats or a rat alarm pheromone. Both conditioning increased rats' freezing to terpinene. Blocking NMDA receptors in the basolateral amygdala prevented odor-specific learning suggesting shock and pheromone-paired pathways converge in the amygdala. An alarm pheromone thus enables cue-specific learning as well as signalling danger.

Figure 1

Conditioned fear can be transmitted to conspecifics in the absence of an external aversive stimulus. (A) Schematics of the odor conditioning and testing paradigm. (B) Percentage freezing time during the testing to the conditioned odor terpinene. (C) Percentage freezing time during the testing to the novel control odor octanol. O⁻/S⁺, shock only rats; O⁺/S⁻, odor only rats that were caged alone; O⁺/S⁺, odor/shock conditioned rats; O⁺/Comp, odor only rats that were caged with odor/shock conditioned rats; O⁺/Comp + Prop: O⁺/S⁻ comp rats that were injected propranolol before training; O⁻/Comp: companion rats without subsequent odor exposure. *p < 0.05, **p < 0.01. Error bars, mean ± SEM.

Figure 2

Pheromone odor conditioning and classical odor conditioning activate distinct but converging circuitries in the brain. (A) An example of Arc and H1a mRNA staining. “1” indicates a double labeled cell (green and red in the nucleus) that was activated by both terpinene and octanol; “2” indicates a cell expressing H1A (green; activated by octanol); “3” indicates a cell expressing Arc (red; activated by terpinene). Scale bars, 100 and 20 µm. (B–K) Ratios of Arc⁺/H1A⁺ cells in various olfactory and limbic structures. O⁻/S⁺, shock only rats; O⁺/S⁻, odor only rats that were caged alone; O⁺/S⁺, odor/shock conditioned rats; O⁺/Comp, odor only rats that were caged with odor/shock conditioned rats. *p < 0.05, **p < 0.01. Error bars, mean ± SEM.

Figure 3

Alarm pheromone mediates the fear learning in companion rats. (A) Schematics of the odor conditioning and testing paradigm. (B) Percentage freezing time during the testing to the odor terpinene. (C) Percentage freezing time during the testing to the odor octanol. O⁺/S⁻, odor only rats that were caged alone; Ph-T, terpinene odor exposed rats that were conditioned with previously identified alarm pheromone molecules; SB-T, terpinene exposed rats that were conditioned with soiled bedding; SB-O, octanol exposed rats that were conditioned with soiled bedding. *p < 0.05, **p < 0.01. Error bars, mean ± SEM.

Figure 4

D-APV bilateral BLA infusions prevents both pheromone odor conditioning and classical odor conditioning. (A) Percentage freezing time during the testing to the conditioned odor terpinene in odor/shock conditioned (O⁺/S⁺) rats, infused with either D-APV or saline. (B) Percentage freezing time during the testing to the conditioned odor terpinene in pheromone molecule conditioned rats (O⁺/Ph), infused with either D-APV or saline. **p < 0.01. Error bars, mean ± SEM. (C) Converging pathways of classical and pheromone fear conditioning in rats. MOB: main olfactory bulb; AOB: accessory olfactory bulb; BLA: basolateral amygdala; CeA: central amygdala; PAG: periaqueductal grey.