77 - 83: Punishment-Induced Fear Modifies the Daily Course of Yawning in Rats

Yawning is seen as a stereotypical action pattern [1] because no environmental input changes the sequence of participating movements, although yawning frequency could be modifiable. There is evidence indicating that this is so, at least in pigtail macaques, specifically Macaca nemestrina [2] and Macaca tonkeana [3], primate species in which the yawn rate increases using instrumental procedures. The opposite, a reduction in yawning frequency, has not been empirically studied [however, please see: 4]; nevertheless, it is known that social factors may inhibit overt yawning, though the motor pattern may still occur.

Contexts in which yawning occurs are varied, ranging from those where a subject is relaxed and/or probably bored [5], through embarrassing social situations, to those which produce anxiety, such as when a person waits for an important appointment [6]. Yawning is often associated with emotional situations, but these comprise a variable number of emotions [7]. Two of them, fear and anxiety, could have been confused in some studies on yawning [e.g. 8]. Fear, a reaction to a dangerous situation which is already real and well defined, differs from anxiety which is the emotional anticipation of an aversive situation difficult to predict and control [7].

In this study, we used a strain of Sprague-Dawley rats that yawns at a high rate (22 yawns/h [9]) and instrumental procedures to investigate: (1) whether yawning behavior can be diminished by punishing it with electric shocks, and (2) whether yawning is a response to fear or anxiety using response-dependent punishment and random punishment, respectively. In addition, we paired an auditory stimulus to the electric shock to see if rats associate one stimulus with the other.

Thirty-one high-yawning (HY) male rats were used at the age of 2 months. Rats were housed 4 per Acrylic cage (46 × 32 × 20 cm) with commercially available food and water provided at libitum, and maintained on a light-dark cycle (light: 07:00-19:00 h), and ambient temperature.

The HY strain was obtained by recording yawning in a sample of 2-month-old male rats, from which the male with the most yawns was crossed with one of his sisters. Then he was crossed with one of his daughters and afterwards the HY strain was maintained by brother-sister mating, selecting HY rats [9].

Yawning behavior was recorded in an experimental cage consisting of a glass cage (21 × 24 × 36 cm) that was open on the top and bottom and placed on a grid floor made of stainless steel rods (1-mm diameter and spaced at 1-cm intervals). Electric shocks (0.6-1.3 mA, 0.5 s) were delivered to the floor by an electric shock generator (60-Hz AC) made at the School of Electronics at the Benemérita Universidad Autónoma de Puebla. A digital multimeter was used to verify the intensity of the shocks on the floor rods. The experimental cage and the grid floor were cleaned before testing each rat. For the auditory stimulus, we used a buzzer (5-cm diameter, 80 dB) which was attached to a wood cage (36 cm high) placed next to the experimental cage. The buzzer was operated manually using a switch and chronometer.

On days of the experiment, a trial started when rats were put singly into the experimental cage, which was on a table in a small room that was illuminated artificially. Yawning frequency was recorded to the nearest minute using a chronometer for 30 min on consecutive days from 14:00 to 16:00 h by 2 observers seated on opposite sides of the table. A yawn was considered as a slow opening of the mouth with inhaling and a more rapid closing and exhaling [10]. One group of 8 rats was observed and yawning recorded for 9 consecutive days with no additional stimulus other than that represented by the experimental cage. Another group of 8 rats was observed and its yawning recorded during 8 consecutive days. The first 3 days the rats received no additional stimulus other than that represented by the experimental cage, but the last 5 days they were presented with 7 buzzer noises (80 dB, 5 s) randomly delivered (for which a table of random numbers was used).

Punishment of yawning behavior consisted of delivering a 80-dB loud buzzer noise for 5 s, paired with an electric shock on the last 0.5 s and according to 2 experimental conditions: (1) Eight rats were observed and yawning recorded during 20 consecutive days. The occurrence of a cumulative number of yawns was followed by a buzzer noise paired with a foot shock. The number represented 20% of the yawning frequency attained the preceding period without buzzer-shock pairings, and was proportionally distributed during the 30 min of observation. In addition, the intensity of the electric shock was increased stepwise at 10-min intervals if yawn frequency surpassed the fixed upper limit of 20%. The intensity of the electric shock was increased to achieve a greater suppression of responding [11]. The buzzer-shock pairings were given over 5 and 4 consecutive days, each period preceded by 3-day periods without punishment, and followed by a 5-day period in which the buzzer noise alone occurred at 30-second intervals. (2) As above, but using 7 rats and with the buzzer-shock pairings delivered randomly.

The yawn rate of each rat was pooled and averaged for each treatment (i.e. days in which either no punishment or buzzer-shock pairings or buzzer noise alone was applied to rats). A repeated-measures ANOVA was used to test for differences amongst treatments and Tukey's HSD test was used to make unplanned comparisons. The data were transformed whenever they did not comply with normality. A logistic regression [12] of the probability of yawn occurrence to the periods of punishment and no punishment together was applied to compare yawn course between response-dependent punishment and random punishment. The average yawn rate of the first period of no punishment served as a reference from which each rat's yawn rate was scored as 1 (above average) or 0 (below average). Exact p values are presented for the reader to assess the strength of the statistical results.

Rats which were put in the experimental cage daily, with no stimulus other than the cage itself, had significantly increased their yawn rate on the last observation day (i.e. day 9) relative to day 1 (paired t test, t₈ = 5.87, p = 0.001; fig. 1). Yawn rate of rats which were presented with a random buzzer noise for 5 consecutive days did not differ from the preceding 3 days when no stimulus was added to the experimental cage (paired t test, t₈ = 1.64, p = 0.146).

ANOVA revealed significant differences amongst the treatments when yawns were followed by buzzer-shock pairings (F_{4, 28} = 5.62, p = 0.002; fig. 2). The post hoc analysis showed that yawn rate increased in the second period of no punishment (p = 0.022) and the period in which the buzzer noise alone was applied (p = 0.004) relative to the first period of buzzer-shock pairings. Yawning increased in the period in which only the buzzer noise was applied relative to the second period of buzzer-shock pairings (p = 0.019).

ANOVA also showed significant differences amongst treatments (F_{4, 24} = 5.41, p = 0.003; fig. 2) when the buzzer-shock pairings were randomly applied to the rats. Yawn rate in the period of buzzer noise alone was greater than in the first period of no punishment (p = 0.012) and the first period of buzzer-shock pairings (p = 0.004).

The odds ratio for days of the first period of punishment and second period of no punishment was higher for yawn-dependent punishment than for random punishment (table 1), although the difference was not significant. The odds ratio for days of the second period of punishment and the period of buzzer noise was also higher for yawn-dependent punishment than random punishment, but the difference was far from being significant (table 1).

The main purpose of this study was to investigate how yawning frequency is affected when male adult rats are exposed to foot shocks in either of 2 conditions: response-dependent punishment or response-independent punishment given at random. Both conditions are expected to distinguish between fear and anxiety, respectively. Secondarily, a buzzer noise was paired with the foot shocks to determine if it lowered yawning frequency as a consequence of the possible association made by the rats between the two types of stimuli. Such association did not occur suggesting that conditioning of yawning, as other stereotyped behaviors, is unlikely.

Since repetitive exposure to the same test situation (e.g. an experimental cage) makes rats settle down, the increase in the overall rate of yawning from the first to the last observation day in the cage suggests that it is associated with relaxation or tranquility. So, as previously noted [13], an increase in yawning frequency seems to be correlated with a decreasing level of stress. However, in this study, when the rats were presented with buzzer noises and hence aroused, yawning frequency did not change and instead followed the daily course of increments. Therefore, the rats either habituated very quickly to the presence of the noises, which is unlikely, or there is a tendency for yawning to increase in response to mild stress [14]. An attempt to maintain arousal cannot be, as suggested by earlier studies [15], a satisfactory explanation as a random loud buzzer noise is already a disturbing stimulus.

The findings indicate that neither of the punishment regimes significantly diminished yawning frequency. This accords with previous studies where the difficulty of preventing [4] or increasing yawning frequency using positive reinforcement procedures was demonstrated [16]. The findings showed that response-dependent punishment prevents yawning from attaining the graded increase which characterizes daily exposure to the same test situation, while random punishment does not modify it. Random buzzer-shock pairings increased yawn rate in the buzzer noise condition relative to the first period of no punishment, and hence resembled what occurred when no aversive stimulus was present; however, yawn-dependent buzzer-shock pairings did not increase the rate. This difference indicates that fear caused by punishing yawning kept the overall rate of responding across testing days constant, although the moderate diminution due to punishment sufficed to reveal that yawning is a delayed response to fear. Anxiety produced by random buzzer-shock pairings, in contrast, did not change the constant increase in yawning throughout the testing days, suggesting that yawning is, in this study, insensitive to random presentation of aversive stimuli.

In summary, yawning is a delayed response to fear produced by response-dependent punishment, and is insensitive to anxiety produced by intermittent punishment. Further experiments will clarify the precise mechanisms whereby rats respond yawning to emotional conditions.