Light matters

Dec 11 2015 Published by under Drug Abuse Science, Drug Fatality

A recent exchange on the Twitter reminded me of an old paper from 1968.

The paper in question is

Scheving LE, Vedral DF, Pauly JE. Daily circadian rhythm in rats to D-amphetamine sulphate: effect of blinding and continuous illumination on the rhythm. Nature. 1968 Aug 10;219(5154):621-2. [PubMed]

Scheving68F1The key takeaway message for me is captured in the first figure (click to embiggen), which represents the percentage of rats that died within 24 h of being injected with either 26 mg/kg (darker line) or 30 mg/kg (dotted line) of amphetamine. The X axis depicts the time of day at which the groups were injected and the bar that forms the X axis indicates when the lights were on (6 am to 6 pm) and off.

As you are aware, rats are a nocturnal species and the wiggle trace just above the X-axis confirms this with activity patterns based on noise recording of the colony.

So, back to the point. The only difference across points within a single amphetamine dose is the time of day at which the drug was administered. Mortality rates change from 20% to nearly 80% with the lowest observed during the inactive part of the rats' day.

Light cycle and circadian phase matter. A lot.

This brings me to a second example, which is from one of the papers in a series of investigations by Dave Roberts at Wake Forest. In

Roberts DC1, Brebner K, Vincler M, Lynch WJ. Patterns of cocaine self-administration in rats produced by various access conditions under a discrete trials procedure. Drug Alcohol Depend. 2002 Aug 1;67(3):291-9. [PubMed]

the authors use a procedure in which rats are allowed to self-administer cocaine 24 h per day. The one major difference from the usual 1-2 h per day type of model is that the number of opportunities for cocaine were limited. These "discrete trial" opportunities ranged from 2-5 per hour and each time the animal was permitted 10 min to make a response once the lever was extended. Each response terminated the discrete trial so animals could only take 2-5 infusion per hour.

Roberts02-coc-circadianThe figure that continues the point most effectively is from a set of manipulations in which the discrete trial was set to 3 per hour and the per-infusion dose was varied. The data represent the total cocaine intake per hour so look at the 1.0 and 2.0 mg/kg/infusion doses if you want to figure out how many responses out of the 3 opportunities per hour were being made.

The point is again obvious, namely that circadian factors and light cycle matter a lot to the outcome. Imagine the more typical 1 h or 2 h operant self-administration session for cocaine being placed at various points across the rat's light cycle. On average, you might expect different mean intakes.

This is going to contribute to replication and reliability issues, particularly if you expect a given mean amount of drug intake.

It gets even tricker if you want to start exploring the effect of different interventions on cocaine self-administration. Who knows if they themselves have circadian-dependent effects or if the interaction with cocaine taking does? Who knows which direction it takes? We don't know until someone does the study.

And we can all see how much exacting work with light cycles there will be to satisfy ourselves that we know what the influence is. Work that, should it turn out negative, will be nigh on unpublishable.

And to be clear, there are hard practicalities of research that make us ignore these factors at times. Mostly across studies, but sometimes within them. Take the big issue of running behavior in the light or dark cycle of a rat (or mouse). This depends on University Facilities level decision making. Can the rooms be reverse-cycled (technically or at the whim of the animal care department)? Can you get access to the right light-cycle room for your animals for your experiments if you are low on the totem pole (as a lab or within a lab)?

Then there are within-lab factors. Limited numbers of operant boxes and limited numbers of hands. You cannot necessarily squeeze all of your animal work into the prime window of 6 h into dark to 12 h identified in the Roberts paper, above. Maybe this function changes depending on your procedures and you have an even narrower stability window.

So there will be compromises.

But these compromises will most assuredly affect the perceived replicability (aka generalization) of the work.

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Additional Reading:

On contradicting your own stuff

The most replicated finding in drug abuse science

12 responses so far

  • Caroline Bass says:

    Funny, I was just looking at the Roberts paper today. To me the most amazing aspect of the discrete trials procedures is that even though the response requirement/effort needed to obtain a moderate dose of cocaine is very minimal, the rats simply don't go after it during the light cycle. So what factors are responsible for this?

    It's been known for a while that a methamphetamine sensitive circadian oscillator exists ("MASCO"), but this hasn't really been studied in terms of motivated behavior.
    One question is whether cocaine self-administration itself is driven by an internal circadian oscillator, if so then the behavior should free-run under constant dim light conditions, which is what we found. So there is an internal "clock" controlling this motivated behavior.

    http://www.ncbi.nlm.nih.gov/pubmed/20524799

    In some ways this isn't surprising, More recently, a paper from Sara Jones' lab did some very elegant work showing how the DAT and dopamine function change with time of day in the striatum.

    http://www.ncbi.nlm.nih.gov/pubmed/24979798

    Most people ignore circadian contributions to behavioral data, but it can have far reaching consequences. Thanks for bringing up such an important subject!

  • Grumble says:

    Your observations are a generalization of the fact that rats and mice did not evolve to interact with an operant box to obtain cocaine, or even food or drink. They evolved to extract nourishment from the environment using behaviors that maximize their reproductive fitness. That includes being nocturnal, but it also includes a lot of other things.

    For instance, something as simple as the size of an animal's meal is dramatically affected by all sorts of parameters, including how much enrichment (i.e., opportunities for doing other things) you give the animal, and how much effort it takes to get the meal.

    One could treat these systematic sources of variability as annoyances on the way to measuring the variable you're really interested in (how much cocaine the animal "wants" to take, how much food the animal "wants" to eat, how much effort the animal "wants" to exert to get it, or whatever). Or, one could realize that these sources of variability are actually what is interesting, in that they tell us something about how the brain does the jobs it evolved to do.

  • drugmonkey says:

    Sure Grumble. But the main point of the day is that *ignoring* such factors and letting that variability creep in and contaminate things is a problem.

  • Caroline Bass says:

    I don't think it's surprising that an animals "desire" to obtain anything is linked to their circadian rhythm and intimately linked with their arousal state. What is motivation if not arousal with direction towards a particular goal? Yet these issues are often ignored. They also have real translational value (addicts tend to overdose a certain times of day, and sleep/arousal/circadian rhythm disruption is a common and difficult withdrawal symptom to deal with). Furthmore, these issues have far reaching implications for the neuroscience underlying these behaviors. Neurons have been known to "switch" phenotypes/subtypes with time of day. Even histology is impacted, if you collect a brain a time of day when your protein of interest is is low amounts it can give you co-localization false-negatives.

    Despite how much behaviorist have adopted the idea of keeping protocols consistent to avoid variability, they still often overlook circadian aspects. For example, collecting brains at the same time of day would be essential when trying to identify a neural substrate underlying the behavior. How many researchers know the time of day their samples were collected?

  • drugmonkey says:

    Or report it accurately?

  • jmz4gtu says:

    Is this why my lingering nicotine addiction never manifests during the daylight hours?

  • shrew says:

    jmz - you're just not hanging out in bars with the bad kids in the daylight hours. Rhythms can be entrained by more than just light, you know.

  • Grumble says:

    "...behaviorists have adopted the idea of keeping protocols consistent to avoid variability..."

    An idea they should perhaps dis-adopt: http://www.nature.com/nmeth/journal/v7/n3/full/nmeth0310-167.html

    See also: http://www.ncbi.nlm.nih.gov/pubmed/25541546

  • Kaleberg says:

    In 'The Youngest Science', Lewis Thomas describes how he learned that penicillin made rabbits' ears go flaccid. He tried to duplicate the result, but failed. His rabbits ears stayed just fine. He figured he had screwed up the protocol somehow, but some years later he read a paper explaining that the ear softening effect was seasonal. It might not just be circadian rhythm.

  • The Other Dave says:

    Doesn't everyone always compare siblings and results obtained within the narrowest possible circadian period?

  • drugmonkey says:

    No. Not everyone does.

  • The Other Dave says:

    That should be listed under 'Rulez of Science', right under 'You cannot keep doing experiments until you get the P-value under 0.05, and then stop'.

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