Many of you may recall that I spent five years in graduate school investigating how alcohol acts on the circadian timing system- behaviorally and physiologically. This was the basis of my dissertation and a research direction that has been pursued by my graduate school mentors for almost a decade. As Drs. Dave Glass and Rebecca Prosser can attest to, it was not an easy journey. Both of them had no formal training in the alcohol field which as we have discovered can be cliquish, placing a HUGE emphasis on pedigree. Hey, I get it. The sleep field is no different and I’m very much part of that pedigree trajectory. However, they both overcame this barrier by proposing and doing fantastic, mechanistic science that has resulted in over 10 original research articles on how long-term and binge-like consumption of alcohol affects circadian timekeeping on many levels.

ResearchBlogging.orgTheir research has combined in vivo (live animal) and in vitro (isolated tissue) experiments that have produced overlapping results, strengthening the body of evidence that alcohol severely disrupts circadian timekeeping. And so, let’s go over this decade of findings.

1. One-time and high (binge-like) doses  of alcohol block the ability of behavioral rhythms to adjust/adapt to unanticipated light. The mechanism involves glutamate. 

This hypothesis was first investigated in hamsters which have very robust rhythms of wheel running and are highly responsive and adaptive to unexpected presentations of light. From here, the same experiments were conducted in mice since this is the “preferred” model of biomedical research given the ability to manipulate its genome. Regardless of the species of study, alcohol inhibited the ability of hamsters and mice to either advance (for hamsters) or delay (for mice) their rhythms to light. These experiments were undertaken by my lab mates and me.

Alcohol dose-dependently prevents animals from adapting to unanticipated light

Dr. Prosser’s lab found that the reason alcohol blocks this adaptation to light is because alcohol blocks glutamate signaling which has been known for over 20 years to affect this adaptation to light.

2. Long-term consumption of alcohol destabilizes daily rhythms of activity. 

These experiments were the most intriguing to me because our hamsters and mice could tolerate drinking a 20% (for hamsters) or 15% (for mice) concentrated alcohol solution and ONLY this alcohol solution for months on end. The deleterious effects of alcohol on daily activity rhythms were striking and present early on. It doesn’t take an expert in circadian rhythms to notice differences in the intensity of daily activity levels between these activity records of mice drinking water (bottom) or drinking alcohol (top) for months on end.

Long-term alcohol intake (top) results in sporadic activity

3. Exercise is an effective substitute for alcohol

Our hamsters inspired us to undertake this experiment because they love to run, running anywhere from 3-5 miles every day, and they love to drink alcohol, drinking 50x as much as the average human male every day. So what we did was to give these “booze-soaked fur balls” access to alcohol in the presence of a locked or unlocked running wheel. When the wheel was unlocked, they ran more and drank less. When the wheel was locked, they drank more. They were also extremely cantankerous during this time, making it very difficult to clean their cages.

Hamsters who run more, drink less

4. Genetic disruption to circadian rhythms (via Per2) increases binge-like, compulsive drinking. 

This work was the most time-consuming for me for several reasons. First, it involved the study of drinking in over 300 mice across 3 years and many, many rounds of microdialysis collection which is one of the more physiologically revealing techniques in neuroscience, but yet very cumbersome because of the low success rate. However, largely thanks to the fact that Kent State University does not charge per diem for animal care, I was able to breed many animals at once to complete this experiment in a timely manner. While this may have taken 3 years at Kent, per diem at most universities could have pushed this project to take 6 years to complete. At any rate, we found that the reason that mice lacking Per2 (in protein form) drink more is because they are awake for two, additional hours during the night and are heavily drinking during this time. This is reflected in the left-side panels of these activity (top) and drinking (bottom) records in comparison to wild-type mice (right-side panels).

Per2 mutant mice are awake more and therefore drink more

5. Acamprosate (market name: Campral) acts in reward-processing and (surprisingly) circadian areas of the brain to reduce alcohol intake

Acamprosate has been on the market for years and yet little attention has been paid to how it acts in the brain. So we used a relatively simple yet under-appreciated and undervalued approach to see where acamprosate acts in the brain. The simplicity resided in mixing up the acamprosate in wax and then popping these tiny wax pellets into the edges of brain areas regulating rewarding behavior and circadian timekeeping. This was complex because these brain areas are relatively small and so our coordinates had to be very precise. Actually, it was through missed targets that we discovered that some brain areas like the hippocampus are not responsive to acamprosate. This figure took over 60 hours to make (seriously) and does a nice job of detailing where in the brain and how effectively acamprosate acts.

Brain areas where acamprosate acts

 

And that’s a wrap. This research direction is far from complete but golly, we sure made an impact on the alcohol field in just ten short years.

Prosser RA, & Glass JD (2014). Assessing ethanol’s actions in the suprachiasmatic circadian clock using in vivo and in vitro approaches. Alcohol (Fayetteville, N.Y.) PMID: 25457753