The Effect of Theophylline on Anxiety in Zebrafish (Danio rerio)Megan Conahan* and Dr. Wendy Boehmler

Department of Biological Sciences, York College of Pennsylvania

Introduction A1 and A2A antagonists have been found to be protective against neurodegenerative diseases.

Compounds that have this property are considered neuroprotective. Methylxanthines, such as caffeine and theophylline are nonselective antagonists of the A1 and

A2A adenosine receptors (Fredholm et al. 1999). Blockade of these receptors has been associated with reduced risk of Alzheimer’s disease and

Parkinson’s disease. Both, theophylline and caffeine have been linked to reduced risk of Parkinson’s disease (Xu et al.

2010). Caffeine has also been found to reduce the risk of Alzheimer’s disease (Dall’Igna et al. 2003)

Caffeine has been found to cause increased anxiety levels by many different studies (Egan et al. 2009). This is an unwanted side effect, suggesting a potential drawback in using caffeine as a neuroprotective agent.

The effect of theophylline on anxiety has been studied very little. It was found in mice that theophylline had a much less potent effect on anxiety (Lopez-Cruz 2014). This suggests that theophylline could be a potential alternative neuroprotective agent.

Zebrafish are increasingly being used as a behavioral model organism, specifically in the field of stress and anxiety. They have also been used to study: social behavior, addiction, sleep, learning and memory. Using zebrafish as a model for these behavioral tests has made them a good candidate for accelerated drug discovery. Other advantages of using zebrafish as a model system include, that they are inexpensive and easy to house in large numbers.

Hypothesis• We predicted that theophylline would increase anxiety in zebrafish, but, have a weaker

effect when compared to caffeine making theophylline an alternative neuroprotective agent to caffeine.

MethodsPre-treatment (n=10)

10 minutes

40 zebrafish

Pre-treatment of plain tank water

(Negative Control)

0.5 mM Caffeine(Positive Control)

0.5 mM Theophylline(Low Dose)

5.0 mM Theophylline(High Dose)

Acclimation Time(2 minutes)

Behavioral Testing(5 minutes)

Results

Negative Control

Positive Control

Low Dose

High Dose

0

2

4

6

Nu

mb

er o

f E

ntr

ies

Figure 1. Mean number of entries into the upper portion of the tank by fornegative control fish (distilled water), positive control fish (0.5 mM caffeine),fish treated with a low dose of theophylline (0.5 mM) and fish treated with ahigh dose of theophylline (5 mM) Sample sizes were n=10 for eachtreatment group. Error bars represent the SEM. According to aKruskal-Wallis test, there was a significant difference across treatmentgroups. (p= 0.0054). Further analysis by post hoc Dunn's multiplecomparison's test found the low dose and high dose of theophylline to besignificantly different compared to the positive control. *p< 0.05, **p<0.01

** *

Tim

e (

%)

Negative Control

Positive Control

Low Dose

High Dose

0

10

20

30

40

50

Figure 2. Mean percentage of time spent in upper portion of tank fornegative control fish (distilled water), positive control fish (0.5 mMcaffeine), fish treated with a low dose of theophylline (0.5 mM) and fishtreated with a high dose of theophylline (5 mM) Sample sizes were n=10for each treatment group. Error bars represent SEM. A Kruskal-Wallis testindicates that means are significantly different (p=0.0028). Furtheranalysis by posthoc Dunn's multiple comparisons test found that both thelow dose and the high dose of theophylline were significantly differentfrom the positive control. *p< 0.05, **p< 0.01.

*

**

Negative Control

Positive Control

Low Dose

High Dose

0

100

200

300

400

Tim

e (s

eco

nd

s)

Figure 3. Mean amount of time to reach upper portion for negative controlfish (distilled water), positive control fish (0.5 mM caffeine), fish treatedwith a low dose of theophylline (0.5 mM) and fish treated with a high doseof theophylline (5 mM) Sample sizes were n=10 for each treatment group.Error bars represent SEM. A Kruskal-Wallis test indicated there was asignificant difference across treatment groups (p= 0.0068). According toa posthoc test, the low dose and high dose of theophylline weresignificantly different from the positive control. *p<0.05, **p<0.01.

**

Conclusions• Caffeine significantly increased anxiety levels compared to

theophylline • The low dose and high dose of theophylline produced significantly

less anxiety than caffeine• This suggests that theophylline would be a good alternative

neuroprotective agent when compared to caffeine• Due to the high through put potential of this model system, it will be

of great interest to continue to identify and test candidate drugs that can be used as neuroprotective agents.

Literature Cited• Dall’Igna, O.P., Porciuncula, L.O., Souza, D.O., Cunha, R.A., Lara, D.R. 2003. Neuroprotection by caffeine

and adenosine A2A receptor blockade of β-amyloid neurotoxicity. British Journal of Pharmacology. 138: 1207-1209

• Egan, R.J., Bergner, C.L., Hart, P.C., Cachat, J.M., Canavello, P.R., Elegante, M.F., Elkhaya, S.I., Bartels, B.K., Tien, A.K., Tien, D.H, Mohnot, S., Beeson, E., Glasgow, E., Amri, H., Zukowska, Z. Kalueff, A.V. 2009. Understnading behavioral and physiological phenotypes of stress and anxiety in zebrafish. Behavioural Brain Research. 205: 38-44

• Fredholm, B.B., Battig, K., Holmen, J., Nehlig, A., Zvartau, E.E. 1999. Actions of Caffeine in the Brain with Special Reference to Factors That Contribute to Its Widespread Use. Pharmacological Reviews. 51: 83-133

• Lopez-Cruz, L., Pardo, M., Salamone, J.D., Correa, M. 2014. Differences between the nonselective adenosine receptor antagonists caffeine and theophylline in motor and mood effects: Studies using medium to high doses in animal models. Behavioural Brain Research. 270: 213-222.

• Xu, K., Xu,Y.H., Chen, J.F., Schwarzschild, M.A. 2010. Neuroprotection by caffeine: Time course and role of its metabolites in the MPTP model of Parkinson Disease. Neuroscience. 167: 475-481.

AcknowledgementsI would like to thank Dr. Wendy Boehmler and Dr. Bridgette Hagerty for their assistance and advice throughout this project.

*This was a blind study

http://en.wikipedia.org

(Egan et al. 2009)

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