Date Published: June 12, 2019
Publisher: Public Library of Science
Author(s): Vincent Bombail, Nathalie Jerôme, Ho Lam, Sacha Muszlak, Simone L. Meddle, Alistair B. Lawrence, Birte L. Nielsen, Patrizia Campolongo.
Most associative learning tests in rodents use negative stimuli, such as electric shocks. We investigated if young rats can learn to associate the presence of an odour with the experience of being tickled (i.e. using an experimenter’s hand to mimic rough-and-tumble play), shown to elicit 50 kHz ultrasonic vocalisations (USVs), which are indicative of positive affect. Male, pair-housed Wistar rats (N = 24) were all exposed to two neutral odours (A and B) presented in a perforated container on alternate days in a test arena. Following 60s of exposure, the rats were either tickled on days when odour A (n = 8) or odour B (n = 8) was present, or never tickled (n = 8). When tickled, rats produced significantly more 50 kHz USVs compared to the days when not being tickled, and compared to control rats. The level of anticipatory 50 kHz USVs in the 60s prior to tickling did not differ significantly between the tickled and control rats. As a retrieval test following the odour conditioning, rats were exposed successively in the same arena to three odours: an unknown neutral odour, extract of fox faeces, and either odours A or B. Compared to controls, 50 kHz USVs of tickled rats increased when exposed to the odour they had previously experienced when tickled, indicating that these rats had learned to associate the odour with the positive experience of being tickled. In a test with free access for 5 min to both arms of a T-maze, each containing one of the odours, rats tickled with odour A spent more time in the arm with this odour. This work is the first to test in a fully balanced design whether rats can learn to associate an odour with tickling, and indicates that positive odour conditioning has potential to be used as an alternative to negative conditioning tests.
Aversive conditioning, where a previously neutral stimulus or place becomes associated with an aversive experience, can be used to study memory and other brain functions in laboratory rodents . This paradigm is used in studies of learning, as odours can be associated with aversive states such as fear [2,3] and malaise [4,5]. The animals usually learn this association very quickly, making it a time-saving and efficient research method, which may be why only few attempts have been made to develop tests for this purpose using positive experiences. In studies where positive conditioning of odours has been applied, they involved pairing with psychostimulant drugs [6–8], alcohol  or a food source [10–12]. However, using feed as the unconditioned stimulus is not always feasible in practice, is likely to be associated with an increasing level of satiety, and psychoactive drugs alter mood and cognition .
All tickled rats emitted significantly more 50 kHz USVs during the sessions with the Tickled procedure than during the sessions when Not Tickled (F2,89 = 31.3; P<0.001), with the latter not differing in magnitude from that of the never tickled Control rats (Fig 2A). This was evident already during the very first tickling session, but with significantly more 50 kHz USVs emitted during the 5th compared to the 1st tickling session (233 vs 83 (±8.4) USVs/min; P < 0.001), and these were significantly correlated (Pearson’s r = 0.53; P = 0.036), indicating that response level of 50 kHz USVs to tickling is a characteristic of the individual rat. No differences in USV frequency were found between rats tickled with Odours A and B, respectively (235 vs 230 (se = 11.7) USVs/min; F1,28 = 0.8; P = 0.383). On days when the tickled rats were Not Tickled (i.e. A-tickled rats when odour B was present and vice versa), the 50 kHz USVs emitted per minute by the 5th tickling session was higher than that found in the 1st session and did not differ significantly from the level observed during tickling in the 1st session, indicating a degree of place association had developed (Fig 2A), whereas the control rats did not show a significant increase with session. Using an appetitive conditioning method, we aimed for rats to learn to associate the presence of an odour with the positive experience of tickling. Our first hypothesis was that rats, which had learned to make the odour-tickling association would emit more anticipatory USVs when exposed to the odour prior to being tickled. This was not the case, as no differences in anticipatory USVs were found between the treatment groups. Indeed, our use of the term anticipatory can be questioned, given the findings. Rats will emit 50 kHz calls in anticipation of access to a running wheel . Others have found that individual rats vocalize more in a chamber associated with play than in a habituated control chamber , indicative of anticipation of positive experiences. The absence of similar anticipatory vocalisations in the present experiment would indicate that, with respect to our first hypothesis, the conditioning paradigm was not successful. However, the levels of USVs emitted during the (anticipatory) pre-session minute appeared to predict the overall level of vocalisation for individual rats when these were not tickled, suggesting that rats can be categorised according to their USV frequency independent of any tickling occurring. This is in accordance with [39,42], who divergently selected rats based on their 50 kHz vocalisations. It has previously been found that tickling-induced 50 kHz ultrasonic vocalizations are individually stable and can predict behaviour in tests of anxiety and depression in rats [43,44]. Our finding that USVs produced when not being tickled show large inter-individual differences, but little intra-individual variation, is complementary to these results. Source: http://doi.org/10.1371/journal.pone.0212829