What is Caffeine? 

Caffeine is considered to be one of the most consumed substances in the world, something that many individuals rely on to feel more alert and awake. Caffeine is a substance that helps people feel more awake and alert. Caffeine is also found to increase endurance, intermittent, and resistance, exercise in humans.¹ Currently in the United States, about 89% of adults consume caffeinated beverages on a daily basis.² Caffeine is almost found in all products, such as coffee, tea, soft drinks, energy drinks, energy bars, some chocolate, non-prescription medication, and much more. ³ Caffeine is able to keep people from feeling tired and have them feel more awake and more aroused because caffeine is a psychoactive compound that blocks the adenosine receptors in the brain. The adenosine receptors (A_2ARs) are found in many different areas of the  brain that include the striatum, olfactory bulb, and other places.⁴ However, adenosine receptors are found to be widespread throughout the brain and that is because adenosine is essential neuromodulator molecule in the brain.⁵ There are three other adenosine receptors, but adenosine 2A receptors (A_2ARs) are responsible for wakefulness and arousal and adenosine 1A receptors (A_1ARs)  are responsible for sleep.⁵ Since, caffeine blocks the adenosine receptors, it inhibits the release of  GABA. GABA is responsible for inhibiting neurons such as dopamine that are involved in wakefulness and arousal. GABA stops the signal or connection from even happening. Caffeine is the antagonist in this action. Caffeine helps increase the production of dopamine.⁶ 

In a paper that was published in The Journal of Neuroscience by Michael Lazarus and colleagues investigated whether or not removing the adenosine A2A receptors (A_2ARs) from the nucleus accumbens (NAc) will affect how caffeine effects wakefulness or arousal.⁷ This was done by looking at the activity and EEG and EMG of three different genetic mouse lines. Global A_2AR  knock-out mice (A_2AR KO), which are mice that do not have a functioning Adora2a gene.^7,8 Basal ganglia-A_2AR knock-out mice (BG-A_2AR KO) are mice that do not have a functioning Adora2a gene within the basal ganglia.⁷ They lastly looked at mice with a loxP-site inserted A_2AR gene, which are mice that have a lox-P site that have the A_2AR gene added to  them.⁷ LoxP-site-inserted is known as a DNA modification that allows researchers to target one specific area.⁹ By using these three different genetic mouse lines, the researchers were able to target and look at a specific are of the brain to see the importance of the adenosine A2A receptors (A_2ARs). 

The Experiment 

In order to figure out whether or not removing the adenosine A2A receptors (A_2ARs) from the nucleus accumbens (NAc) would affect wakefulness and or arousal with caffeine, mice were used. There were three different genetic mouse lines that were used in this study, a global A_2AR knock-out mice (A_2AR KO), a basal ganglia-A_2AR knock-out mice (BG-A_2AR KO) which are mice that lack the BG-A_2ARs and mice with a loxP-site-inserted A_2AR gene that is amenable to conditional disruption by the injection of Cre recombinase-expressing AAV. These three give the genetic mouse models that were used throughout the study. The researchers then did a vigilance state assessment using electroencephalogram, electromyogram and locomotor activity reading. This was first performed on the A_2AR KO mice, and, under anesthesia, the mice were implanted with the electroencephalogram (EGG) and electromyogram (EMG) electrodes for the  reading on the frontal cortical area. The EMG activity was then monitored, and the mice were placed into experimental cages for an acclimatization period after a recovery period. The other two genetic mice were also put through the same procedures. On the first day, all the mice were treated with vehicle (saline solution) at the same time and the EEG and EMG signals were collected for 24 hours. Then, on the second day, the mice were treated with caffeine and EEG  and EMG signals were collected once again for 24 hours. The samples were then put through a  SLEEPSIGN software and digitalized and amplified. The locomotor activity was also recorded with the help of an infrared photo center cell. The vigilance states were scored in three different stages, waking rapid-eye movement (REM) sleep and non-REM (NREM) sleep. Then the assessment of activity and inactivity were also performed. This was done by injecting the mice with either vehicle or caffeine. Then activity and inactivity were assessed based on sleep and wakefulness. Then a generation of AAV vectors were performed on a set of new mice and the AAV injections were performed. After the injection the mice went for implantation of EEG and EMG. Caffeine or vehicle injections were performed. Then the mice were later deeply anesthetized, and their brains were removed and sectioned off in sections. The sections were then scanned into program and statistical analysis was also completed. 

Findings

The findings from the study found that the deletion of the adenosine receptors (A_2ARs) in the basal ganglia of mice saw that the mice did not have any wakefulness or arousal effect on caffeine.⁷ The researchers also found that there were other A_2ARs in cell groups that were surrounding parts of the striatum. The striatum is known as an area of the brain that part of the reward system and also known for receiving glutamatergic and dopaminergic inputs from different areas of the brain.¹⁰ When the mice were injected with the caffeine, the basal ganglia A_2AR knock-out mice (BG-A_2AR KO) was the only one that did not have the wakefulness or arousing from caffeine. This could mean that the A_2ARs in the basal ganglia play a role in the process of wakefulness and arousal and along with that also sleep. It was also found in the study that the deletion of the adenosine receptors (A_2ARs) in the basal ganglia of mice also saw less caffeine-induced locomotor activity.⁷ This just means that the mice had no changes in activity and were not either hyperactive or hypoactive, and no changes in their sleep. The study found that the selective deletion of the adenosine receptors (A_2ARs) in the nucleus accumbens (NAc) shell eliminates the arousal and wakefulness when on caffeine.⁷ Whereas, the mice that had no deletions saw that caffeine did affect both the arousal and wakefulness and locomotor activity.⁷ These mice were also seen to have different REM and NREM sleep cycles and more brain activity shown in the EEG and EMG. The findings also showed that the nucleus accumbens (NAc) shell is conditioned to be the main regulation for sleep. The researchers were able to contain and pinpoint the activity of the adenosine receptors (A_2ARs) and found that the nucleus accumbens (NAc) contains a high density of the receptors. It was also seen that caffeine has the power to override the adenosine projections to promote wakefulness. This shows that adenosine is there to help regulate the homeostasis of sleep.

Concluding Remarks 

This study shows how important the adenosine receptors (A_2ARs) are and how the receptors play a major role in wakefulness and arousal. However, it also showed how too much caffeine can affect the body's regulation of sleep. The adenosine receptors (A_2ARs) were seen to be in all areas of the brain. More studies on the adenosine receptors (A_2ARs) areas still need to be conducted. This study could be used for future studies testing on drugs that have the similar effect as caffeine. If more is known on how caffeine affects the brain, then newer products could be developed so that people are not so dependent on just caffeine. This study could also be used to show how drinking caffeine late at night can affect the brain to not be able to maintain and regulate sleep. Which in the long run could have negative consequences for not getting adequate sleep. A longer study on caffeine could also show whether or not too much caffeine is harmful to the brain and its activity. 

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