Caffeine is found in many beverages (such as tea, coffee, cocoa and cola), foods (such as chocolate) and also used therapeutically (Bhattacharya, Satyan & Chakrabarti, 1997; Daly & Fredholm, 1998). Caffeine affects the central nervous system, as it is an adenosine antagonist that has been found to readily cross the blood brain barrier, through diffusion and carrier-mediated transporters (Mc Call, Millington & Wurtman, 1982). As caffeine is an adenosine antagonist, it has an opposing effect to adenosine (Garrett & Griffiths, 1997). That is, adenosine produces depressant effects, whereas caffeine has stimulating effects. Adenosine acts on both A1 and A2A receptors, which are involved in the regulation of motor activity (Antoniou et al., 2005). Caffeine increases locomotor activity by blockade of adenosine A2A receptor (Yacoubi Ledent, MeArnard, Parmentier & Vaugeois, 2000). Furthermore, caffeine has also been found to have a biphasic effect on animals and humans (Daly & Fredholm, 1998). Thus, taken in low doses caffeine increases locomotor activity and in high doses decreases it.
Bolles (1960), suggests rats normal grooming behaviour, is one third of their day, with a higher incidence after eating and drinking and just before they go to sleep in the evening. Caffeine has been found in open field behaviour studies on rodents, to increase grooming, ambulation, rearing, sniffing and sniffing air, and wheel-running, whilst decreasing immobility (Antoniou et al., 2005; Davis, Zhao, Stock, Mehl, Buggy & Hand, 2002; Meliska & Loke, 1984; Uzbay, Kose, Kayir, Ulusoy & Celik, 2009; Yacoubi et al., 2000). In contrast, Bhattacharya, Satyan and Chakrabarti (1997) found caffeine to reduce ambulation and rearing, but increased immobility and defaecation. Uzbay and associates (2009) found no gender differences in locomotor activity of mice. Furthermore, caffeine has been found to increase anxiety in rats, including caffeine-induced anxiety and withdrawal anxiety in chronic caffeine users (Bhattacharya, Satyan & Chakrabarti, 1997). However, in human investigations, Lieberman and associates (1987) found caffeine improved reaction time and vigilance (including both visual and auditory), with no increase in tension and anxiety.
As caffeine is consumed by many people through the various different forms, it is important to investigate the effects it may have. Thus far, research has been contradictory in the outcomes from the aforementioned studies. Furthermore, most of the studies (excluding Uzbay et at., 2009; Lieberman, Wurtman, Emde, Roberts & Coviella, 1987) have only investigated male rodents. Therefore, it is the hypothesis of the present study that caffeine will increase ambulation, rearing, defaecation, sniffing and grooming. The present study will also search for gender differences between the experimental (caffeine) and control group.
Six male and six female 120 day old, Long-Evans hooded rats were used. The rats were maintained on a 12-h light/dark cycle with food and either water or the caffeine solution available ad libitum, and with a constant ambient temperature of 23.3oC.
The rats were randomly assigned, through the use of a random numbers table, to the experimental or control group, with the only restriction being that an equal number of male and female rats were assigned to each group. Two 10 litre containers of saline, one with only saline and the other consisted of caffeine (2g/litre) dissolved in the 10 litres of saline, was prepared, coded and stored at 4oC. The rats were then given their assigned saline or caffeine solution ad libitum for two days. The observer was blind to the gender and treatment condition of the rats, to restrict observer bias.
Apparatus and procedure
The experiment was conducted in a well-illuminated 15m x 30m laboratory, with suspended lights, and a wide variety of apparatus stored along the walls. The room remained a constant, ambient temperature of 23.3oC throughout the experiment. The behaviour experiments were conducted between 08:00 and 10:00 h on the third day. The test chamber was a transparent plastic open field cage (44.5 x 44.5 x 30 cm), with a white floor. The chamber was monitored by a Quasar video recorder suspended directly above the chamber. Prior to recording the behaviour, a 15 second habituation period was given. Behaviour was recorded for 3 minutes and fifteen seconds, and the frequency of each behavioural response was recorded for the entire duration. The floor was divided into a 3 x 3 matrix, by lines drawn on the computer screen (screen size 17 inch), at equal intervals (4.5cm x 4.5cm). The following behavioural responses were recorded: ambulation, which was recorded by when the rat crossed over and entered a square with all four paws, if only the forepaws entered the square it was not recorded; rearing, body inclined vertically, with both hind paws remained on the floor, with both forepaws off the floor, including when they were on the side walls; defaecation, by total faecal pellets on the floor at the end of the recording; sniffing, which was recorded when the rat was not moving and sniffing the floor, and also when the rat was sniffing the walls; grooming, which was recorded when the rat cleaned it's face or any other part of its body.
All data was analysed by one-way analysis of variance, followed by two-sample assuming equal variances t-test. The levels of statistical significance were set at p < .05. Statistical significance is presented in results and in tables.
Specific behavioural measures
(Data are summarized in table 1 and figures 1, 2 and 3).
Statistical analyses revealed that caffeine treated subjects produced a statistically significant increase in the number of squares entered and crossed in the open-field (M = 40.33, SD = 13.11), t(10) = 3.56, p < .05 (Figure 1). Significant effects were also observed for gender differences between the caffeine and control groups, males t(4) = 4.89, p < .05 and females t(4) = 3.81, p < .05. However, there were no significant effect between the male and female caffeine group t(4) = 2.62, p > .05.
The frequency of rearing was increased in the caffeine treated subjects, producing a statistically significant increase in frequency (M = 25.16, SD = 10.0), t(10) = 2.408, p < .05 (Figure 2). However, there were no significant effects observed for gender differences between the caffeine and control groups, males t(4) = 2.61, p > .05 and females t(4) = 2.06, p > .05. There was also no significant effect between the male and female caffeine group t(4) = 1.82, p > .05.
Frequency of defaecation was decreased in the caffeine treated subjects, revealing no statistical significance (M = 1.33, SD = 1.63) t(10) = 1.509, p > .05. There was a statistical significant effect between the caffeine and control group males t(4) = 3.61, p < .05. However, there was no significant effect between females. There was also no significant effect between the male and female caffeine groups t(4) = 1, p > .05.
The frequency of sniffing was increased in the caffeine treated subjects, producing a statistically significant increase in frequency (M = 68.83, SD = 7.02), t(10) = 6.414, p < .05 (Figure 3). There was a statistical significant effect between caffeine and control group males t(4) = 4.81, p < .05 and also between females t(4) = 4.45, p < .05. However, there was no statistical significant effect between the male and female caffeine groups t(4) = 1.3, p > .05.
The frequency of grooming was decreased in the caffeine treated subjects, revealing no statistical significance (M = 1.16, SD = .98), t(10) = 1.83, p > .05. Statistical analyses revealed that caffeine treated female rats decreased their grooming, producing a statistical significant effect t(4) = 7, p < .05. However, there was no significant effect between males in the caffeine and control group. There was also no significant effect between male and female caffeine groups t(4) = 1.34, p > .05.
This study examined whether caffeine increased ambulation, rearing, defaecation, sniffing and grooming in caffeine induced rats, whilst also investigating for gender differences. The findings of the present study indicate that caffeine increased ambulation, rearing and sniffing in rats, which is consistent with previous research (Antoniou et al., 2005; Davis et al., 2002; Meliska & Loke, 1984; Uzbay et al., 2009; Yacoubi et al., 2000). Previous studies suggested defaecation and grooming are also increased after caffeine was administered (Antoniou et al., 2005; Bhattacharya, Satyan & Chakrabarti, 1997). However, the present study found defaecation and grooming, decreased in caffeine induced rats. This could be explained from methodological differences. For example, in the present study the rats self-administered the caffeine, whereas previous investigations have utilized intraperitoneal administration (Antoniou et al., 2005; Bhattacharya, Satyan & Chakrabarti, 1997). Another explanation could also be attributed to the different species and strains of the animals used. For example, the present study utilized Long-Evans hooded rats, whereas others have used different strains of rats (Bhattacharya, Satyan & Chakrabarti, 1997) and different species, such as mice (Antoniou et al., 2005). A third explanation could also be the individual differences between the rats in their normal behaviour. As Bolles (1960), found some rats groomed more than others. Furthermore, the finding in the present study for increased ambulation and rearing could be due to other factors such as anxiety (Bhattacharya, Satyan & Chakrabarti, 1997) and fear (Meliska & Loke, 1984).
The use of comparing genders on five different behavioural measures has provided an increased understanding of the different effects caffeine can have on the different genders. For example, the present results found a statistically significant difference in grooming between female rats, with the caffeine induced rats decreasing their grooming behaviour. Furthermore, there was a large statistical significance between the caffeine and control group males on ambulation, although there were no significant effects found between the genders in the experimental group, which suggests caffeine affects both genders in much the same way, dependent on what is normal for their gender. This is consistent with previous findings, even though different species were used (Uzbay et al., 2009). The gender differences found in the present study could also be explained from the different hormones that may be affected from the caffeine, which requires further investigation.
Moreover, the effects found in the present study, although consistent with previous findings for ambulation, rearing and sniffing, utilized different methodology. A limitation in the present study is that the behaviour was recorded for a shorter duration than previous investigations, which recorded for up to one hour (Antoniou, et al., 2005). Uzbay et al., (2009), found locomotor activity in mice reached the maximum level within 30 minutes. Which suggests that behaviour recording for a longer duration than the present study, would be beneficial for more accurate results.
Another limitation in the present study was that there were no measures taken for the accuracy of the amount of caffeine ingested. Therefore, it is difficult to be confident in the behavioural effects from ingesting caffeine. Future investigations could benefit from using different techniques, such as intraperitoneal administration or housing the rats in separate cages so that more accurate measures of caffeine intake can be recorded. Future investigations could also benefit from recording behaviour at different intervals throughout the day, as the present study only recorded behaviour in the morning and different behaviours may arise at different times. Bolles (1960) found rats increased grooming in the evening, and as the present results found grooming was decreased in the caffeine induced group, it would be beneficial to also record the behaviour in the evening. Another limitation in the present study is that a small sample was used, future investigations may benefit from utilizing a larger sample to increase the accuracy of the results.
In summary, it can be concluded that caffeine increases hyperactivity in rats' behaviours of ambulation, rearing and sniffing. As caffeine is a psychoactive drug that can increase stimulation by blocking adenosine receptors (Yacoubi et al., 2000), it may be beneficial for people who require extra stimulation, such as prior to exercising or competing in sport.