Stability of 50-kHz ultrasonic response: male and female

male and female high and low chirpers

In all experiments, the gradual rise in the number of 50-kHz chirps over repeated tickling sessions stabilized on an individually specific level by the second week of manipulation (Figures 16 and 17 and Paper V). The average 50-kHz USV response to daily tickling on days 12–14 proved to be a good predictor of the subsequent USV response. Rats divided into groups of high and low chirpers (HC and LC, respectively) by the median split of this measure revealed respectively high and low levels of 50-kHz USVs both over prolonged periods of continued tickling as well as after a pause in tickling sessions and regardless of housing conditions (single- or group housing) and sex. Also, a significant between-days correlation of the 50-kHz responses on different test days was found in the first week already, but this response did not predict the animal’s chirping levels on the subsequent weeks. Similar pattern of change over repeated tickling sessions was visible in both sexes in the FM 50-kHz USVs (Figure 17), suggesting that at least in the context of daily experimenter-induced stimulation in juvenile rats there is no qualitative difference between the flat and FM USVs as a response to this stimulation. The levels of FM calls were somewhat lower in both sexes as compared to the flat USVs at baseline levels, while after social housing this difference was visible in male rats only.

Previously, Burgdorf and Panksepp (2006) have reported more trill-type USVs than flat USVs in response to tickling in adult females and that with regard to the flat-type USVs the high-chirping rats do not differ from the low-chirping group. It seems reasonable to assume that differences in experimental design stand behind these variations, suggesting that if the tickling procedure is started when the animals have reached adult age, the resulting USV profile may differ to a certain extent from the conditions where tickling sessions have been started at weaning already. Age-related decreases in USV response to tickling have been reported (Panksepp and Burgdorf, 2003), and in the light of the above-mentioned results it may be suggested that the flat-type 50-kHz USVs show a greater and earlier decrease over aging process that may be related to different relevance and „meaning“ of the two types of USVs at different age points.

The animals were always single-housed during the tickling sessions over the first two weeks since the 50-kHz response to tickling has been shown to be greater in such conditions (Panksepp and Burgdorf, 2000). The number of tickling-induced 50-kHz USVs decreased over periods of social housing that has been found to reduce tickling-induced USVs already in 48 h after moving isolate-housed animals to group housing (Burgdorf and Panksepp, 2001), but

the differences between HC and LC animals were retained. It is noteworthy that the levels of FM chirps did not decrease in females over the period of social housing, and the decrease in flat USVs was not significant in female LC animals. This suggests that at least in LC females social housing has a different effect on USVs than in other groups (and more so on FM chirps), that seems a reasonable assumption in the context that females have been found to have different social behaviour profiles than males (Douglas et al., 2004; Pellis et al., 1997), and remain playful longer after puberty as compared to males (Panksepp et al., 1984).

Figure 16. The levels of responding to tickling (four 15 s sessions of tickling inter-mittently with 15 s pauses) with 50-kHz chirping in male (A) and female (B) rats over 41 days of treatment, with a pause in tickling between Days 21–40 (Paper V). The animals were single-housed over the first 21 days of the Experiment, and group-housed by 4 after that. n = 6, 9, 10, 10(male HC and LC; female HC and LC, respectively). The animals were divided into groups with high and low levels of 50-kHz USVs by median split of the average result of Days 12–14 of tickling. Significant differences between the male groups: Days 3, 7, 12, 15–18 p<0.05; Days 9, 11, 14 p<0.01. Significant differences between the female groups: Days 3, 6, 20, 41 p<0.05; Days 7, 8, 9, 16, 17, 19, 21, 40 p<0.01; Days 11, 12, 15, 18 p<0.001; Days 13, 14 p<0.0001. HC – high chirping rats; LC – low chirping rats; Day – day of tickling (zero equals to the day of weaning). Data are presented as mean ± SEM.

Figure 17. Ultrasonic vocalizations in male (A) and female (B) rats before and after chronic stress (Paper VI). * – p<0.05; ** – p<0.01; *** – p<0.001 vs respective LC; # – p<0.05 vs respective stress. The animals were divided into groups with high and low levels of 50-kHz USVs by median split of the average result of Days 12–14 of tickling.

HC – high chirping rats; LC – low chirping rats. Data are presented as mean ± SEM.

We also studied the emission of low-frequency USVs in the range of 20 kHz, that have long been associated with negative events and stimuli (Vivian and Miczek, 1991). In order to learn whether the 22- and 50-kHz USVs and hence the emotional messages they carry are mutually exclusive or not, the as-sociations between the two types of USVs were assessed, and no correlations were found in untreated rats, although some female HC-rats tended to emit increasing levels of 22-kHz USVs over repeated testing (Paper V). Previously, Burgdorf et al. (2005) have found a negative correlation between the two types of USVs in rats that were bred for high levels of 50-kHz chirps. It is possible that there are differences in 22-kHz USV levels between animals from this breeding experiment and from the present first generation studies that play a

significant role in this regard. It has been suggested that the vocalizations and movements made by the tickled subject carry signals of both pleasure and submission, warning that, when ignored by the tickler, the normally pleasant stimulation may become unpleasant (Brudzynski and Ociepa, 1992). This implies that the 22- and 50-kHz USVs should be emitted simultaneously in specific situations, which was true in the present experiments, in which the two types of USVs were detected in identical experimental conditions. Thus, the 22- and 50-kHz USVs are not mutually exclusive, but permit the expression of different aspects of affective states in the animal quite simultaneously.

4.2.2. Behavioural differences in male and