Odour conditioning of positive affective states: Can rats learn to associate an odour with tickling?

Rodents can learn to associate a neutral stimulus with a negative experience, such as an electric shock. Few attempts have been made to create associations with positive experiences. In rats, tickling (i.e. mimicking rough-and-tumble play with an experimenter’s hand) has been shown to elicit 50 kHz ultra-sonic vocalisations (USVs), which have been compared to laughter in humans. We investigated if young rats could learn to associate the presence of an odour with the putatively positive experience of being tickled. Male Wistar rats (N=24) were pair-housed, and habituated to an inverse lighting schedule as well as being transported to a transparent test arena with sawdust litter and an empty container. All rats were placed in the arena on each testing day (10 days in total), and one of the odours (A and B) were present on alternate days. The rats were subjected to one of 3 treatments (n=8) under red light: never tickled (CON), or tickled, only when odour A (TA) or odour B (TB) was present in the container. After 1 min in the arena, tickling began and consisted of four 20 s episodes of tickling interspersed with 3 pauses each of 20 s where the hand was motionless on the side of the arena, which was also where the hand was for CON and the non-tickled rats of the day. Tickled rats produced significantly (P<0.05) more USVs when being tickled (mean 233±9 USVs/min) compared to the pauses (mean 151±8) and the days when not being tickled (mean 59±7), the latter not differing from the USVs produced by the control rats (mean 29±8). The level of anticipatory USVs in the minute prior to tickling did not differ significantly among the TA, TB and CON rats. After the 10-day conditioning, we carried out a 3-odour test in the same arena and set-up, but with no tickling or hand present. Rats were placed in the arena for 30s, and then exposed to 3 odours for one minute each; with 30 s pauses in between. The 3 odours were an unknown odour, extract of fox faeces, and either odour A (for the TA and half of the CON rats) or odour B (for the TB and remaining half of the CON rats). USVs decreased gradually across the testing period, but increased above that of controls for the tickled rats when exposed to their tickling odour (TA: 41±8, TB: 66±11, CON: 20±7 USVs/min), although this only reached significance for odour B (P<0.01). Although the rats did not produce more anticipatory USVs when exposed to their tickling odour, they did increase the USV frequency when the odour was presented following two unknown odours, one of which was assumed to be fear inducing. This finding indicates that the rats had learned to associate an odour with the positive experience of tickling. This was not simply because the odour was known, as the control rats showed no such increase. Positive odour conditioning may thus have potential to be developed further with a view to replace negative odour conditioning tests.